Elementary mechanisms of calmodulin regulation of NaV1.5 ...

Elementary mechanisms of calmodulin regulationof NaV1.5 producing divergentarrhythmogenic phenotypesPo Wei Kanga,b,1,2

, Nourdine Chakouric,1, Johanna Diazc, Gordon F. Tomasellid, David T. Yuea,and Manu Ben-Johnya,c,2

aDepartment of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218; bDepartment of Biomedical Engineering, Washington Universityin St. Louis, St. Louis, MO 63130; cDepartment of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032; and dDepartment ofMedicine, Albert Einstein College of Medicine, Bronx, NY 10461

Edited by Kurt G. Beam, University of Colorado Anschutz Medical Campus, Aurora, CO, and approved April 13, 2021 (received for review December 16, 2020)

In cardiomyocytes, NaV1.5 channels mediate initiation and fast prop-agation of action potentials. The Ca2+-binding protein calmodulin(CaM) serves as a de facto subunit of NaV1.5. Genetic studies andatomic structures suggest that this interaction is pathophysiologicallycritical, as human mutations within the NaV1.5 carboxy-terminus thatdisrupt CaM binding are linked to distinct forms of life-threateningarrhythmias, including long QT syndrome 3, a “gain-of-function”defect, and Brugada syndrome, a “loss-of-function” phenotype.Yet, how a common disruption in CaM binding engenders divergenteffects on NaV1.5 gating is not fully understood, though vital forelucidating arrhythmogenic mechanisms and for developing newtherapies. Here, using extensive single-channel analysis, we findthat the disruption of Ca2+-free CaM preassociation with NaV1.5exerts two disparate effects: 1) a decrease in the peak open proba-bility and 2) an increase in persistent NaV openings. Mechanistically,these effects arise from a CaM-dependent switch in the NaV inacti-vation mechanism. Specifically, CaM-bound channels preferentiallyinactivate from the open state, while those devoid of CaM exhibitenhanced closed-state inactivation. Further enriching this scheme,for certain mutant NaV1.5, local Ca

2+ fluctuations elicit a rapid re-cruitment of CaM that reverses the increase in persistent Na current,a factor that may promote beat-to-beat variability in late Na current.In all, these findings identify the elementary mechanism of CaMregulation of NaV1.5 and, in so doing, unravel a noncanonical rolefor CaM in tuning ion channel gating. Furthermore, our results fur-nish an in-depth molecular framework for understanding complexarrhythmogenic phenotypes of NaV1.5 channelopathies.

Nav1.5 | calmodulin | Brugada syndrome | long QT syndrome | ion channels

Voltage-gated sodium channels (NaV) are responsible for theinitiation and spatial propagation of action potentials (AP)

in excitable cells (1, 2). NaV channels undergo rapid activationthat underlie the AP upstroke while ensuing inactivation permitsAP repolarization. The NaV1.5 channel constitutes the predomi-nant isoform in cardiomyocytes, whose pore-forming α-subunit isencoded by the SCN5A gene. NaV1.5 dysfunction underlies di-verse forms of cardiac disease including cardiomyopathies, ar-rhythmias, and sudden death (3–6). Human mutations in NaV1.5are associated with two forms of inherited arrhythmias–congenitallong QT syndrome 3 (LQTS3) and Brugada syndrome (BrS) (7).LQTS3 stems from delayed or incomplete inactivation of NaV1.5that causes persistent Na influx that prolongs AP repolarization—a“gain-of-function” phenotype (7–9). BrS predisposes patients tosudden death and is associated with a reduction in the peak Nacurrent that may slow cardiac conduction or cause region-specificrepolarization differences—a “loss-of-function” phenotype (10, 11).Genetic studies have identified an expanding array of mutations inmultiple NaV1.5 domains, including the channel carboxy-terminus(CT) that is a hotspot for mutations linked to both LQTS3 andBrS (12, 13). This domain interacts with the Ca2+-binding proteincalmodulin (CaM), suggesting that altered CaM regulation of

NaV1.5 may be a common pathophysiological mechanism (12,14–16). More broadly, human mutations in the homologous re-gions of neuronal NaV1.1 (17, 18), NaV1.2 (19, 20), and NaV1.6(21) as well as skeletal muscle NaV1.4 (22) are linked to variedclinical phenotypes including epilepsy, autism spectrum disorder,neurodevelopmental delay, and myotonia (23). Taken together, acommon NaV mechanistic deficit—defective CaM regulation—may underlie these diverse diseases.CaM regulation of NaV channels is complex, isoform specific,

and mediated by multiple interfaces within the channel (14–16).The NaV CT consists of a dual vestigial EF hand segment and acanonical CaM-binding “IQ” (isoleucine–glutamine) domain(24, 25) (Fig. 1A). The IQ domain of nearly all NaV channelsbinds to both Ca2+-free CaM (apoCaM) and Ca2+/CaM, similarto CaV channels (26–31). As CaM is typically a Ca2+-dependentregulator, much attention has been focused on elucidating Ca2+-dependent changes in NaV gating. For skeletal muscle NaV1.4,transient elevation in cytosolic Ca2+ causes a dynamic reduction inthe peak current, a process reminiscent of Ca2+/CaM-dependentinactivation of CaV channels (32). Cardiac NaV1.5 by comparisonexhibits no dynamic effect of Ca2+ on the peak current (32–34).

Significance

Calmodulin (CaM) regulation of cardiac NaV channels is vital forcardiac physiology and pathophysiology. Channelopathic muta-tions in NaV1.5 that disrupt CaM binding trigger two mechanisti-cally divergent arrhythmia syndromes. Specifically, long QTsyndrome 3 results from a gain-of-channel function, while Bru-gada syndrome stems from a loss-of-channel function. Yet,mechanisms that elicit seemingly paradoxical changes in channelfunction are unknown. Using single-channel analysis, we dem-onstrate that the disruption of CaM binding to NaV1.5 diminisheschannel activity and enhances the propensity for persistent Na+

current, all resulting from a switch in the NaV inactivation mech-anism. These findings reveal insights into the mechanism of CaMregulation of NaV channels as well as inform upon alterations inchannel function that trigger life-threatening arrhythmias.

Author contributions: P.W.K., G.F.T., D.T.Y., and M.B.-J. designed research; P.W.K., N.C.,J.D., and M.B.-J. performed research; P.W.K., N.C., J.D., G.F.T., D.T.Y., and M.B.-J. contrib-uted new reagents/analytic tools; P.W.K., N.C., J.D., and M.B.-J. analyzed data; and P.W.K.,N.C., J.D., G.F.T., and M.B.-J. wrote and revised the paper.

The authors declare no competing interest .

This article is a PNAS Direct Submission.

Published under the PNAS license.1P.W.K. and N.C. contributed equally to this work.2To whom correspondence may be addressed. Email: [email protected] [email protected].

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2025085118/-/DCSupplemental.

Published May 21, 2021.

PNAS 2021 Vol. 118 No. 21 e2025085118 https://doi.org/10.1073/pnas.2025085118 | 1 of 12

PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2

https://orcid.org/0000-0002-5933-545X

https://orcid.org/0000-0002-8809-8684

http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.2025085118&domain=pdf

https://www.pnas.org/site/aboutpnas/licenses.xhtml

mailto:[email protected]

mailto:[email protected]

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2025085118/-/DCSupplemental


https://doi.org/10.1073/pnas.2025085118


Instead, sustained Ca2+ elevation has been shown to elicit adepolarizing shift in NaV1.5 steady-state inactivation (SSI or h∞),although the magnitude and the presence of a shift have beendebated (32, 35). Additional CaM-binding sites have been iden-tified in the channel amino terminus domain (36) and the III-IVlinker near the isoleucine, phenylalanine, and methionine (IFM)motif that is well recognized for its role in fast inactivation (35, 37).However, recent cryogenic electron microscopy structures, bio-chemical, and functional analyses suggest that both the III-IV linkerand the Domain IV voltage-sensing domain might instead interactwith the channel CT in a state-dependent manner (38–43).

Beyond Ca2+-dependent effects, the loss of apoCaM bindingto the NaV1.5 IQ domain increases persistent current (34, 44),suggesting that apoCaM itself may be pathophysiologically rele-vant. Indeed, NaV1.5 mutations in the apoCaM-binding interfaceare associated with LQTS3 and atrial fibrillation (7), as well as aloss-of-function BrS phenotype and a mixed-syndrome phenotypewhereby some patients present with BrS while others with LQTS3(Fig. 1A) (13, 45). How alterations in CaM binding paradoxicallyelicits both gain-of-function and loss-of-function effects is not fullyunderstood, though important to delineate pathophysiologicalmechanisms and for personalized therapies.Here, using single- and multichannel recordings, we show that

apoCaM binding elicits two distinct effects on NaV1.5 gating: 1)an increase in the peak channel open probability (PO/peak) and 2)a reduction in the normalized persistent channel open proba-bility (Rpersist), consistent with previous studies (34, 44). The twoeffects may explain how mixed-syndrome mutations in theNaV1.5 CT produce either BrS or LQTS3 phenotypes. On onehand, the loss of apoCaM association may diminish PO/peak andinduce BrS by shunting cardiac AP. On the other hand, increasedRpersist may prevent normal AP repolarization, resulting inLQTS3. Analysis of elementary mechanisms suggests that thesechanges relate to a switch in the state dependence of channelinactivation. Furthermore, dynamic changes in Ca2+ can inhibitpersistent current for certain mutant NaV1.5 owing to enhancedCa2+/CaM binding that occurs over the timescale of a cardiacAP. This effect may result in beat-to-beat variability in persistentNa current for some mutations. In all, these findings explain howa common deficit in CaM binding can contribute to distinctarrhythmogenic mechanisms.

ResultsNaV1.5 SSI Is Unperturbed by Acute Cytosolic Ca2+ Elevation. A pro-longed increase in cytosolic Ca2+ has been suggested to shift thevoltage dependence of NaV1.5 SSI or h∞ curve; however, it isunknown whether dynamic Ca2+ fluctuations as typical for car-diomyocytes might evoke similar effects. As such, we undertookCa2+ photo-uncaging experiments with a modified voltage pulseprotocol that measures h∞ curves within the same cell just beforeand after Ca2+ uncaging. Before Ca2+ uncaging, increasing theholding potentials (−120 to +30 mV) evoked peak currents ofdecreasing amplitudes because of SSI (Fig. 1 B, Left). Normalizingpeak amplitudes of each pulse by the peak amplitude of the firstpulse yielded a baseline h∞ curve at cytosolic [Ca2+]free of∼100 nM (corresponding to diastolic Ca2+ in cardiomyocytes) asshown by the average h∞ curve obtained from multiple cells(Fig. 1 C, Left). A brief ultraviolet (UV) pulse elicited a step-likeincrease in intracellular [Ca2+]free up to ∼4 μM, beyond the peaksystolic cytosolic Ca2+ levels observed in cardiomyocytes (Fig. 1 B,Right). The h∞ curve obtained following Ca2+ photo-uncaging,however, overlayed with that at low Ca2+ levels (Fig. 1 C, Right).Thus, NaV1.5 SSI appears to be insensitive to transient changes incytosolic Ca2+ as would be observed during each cardiac beat.Absent dynamic Ca2+ effects, we reasoned that the apoCaM

interaction may be more consequential for NaV1.5 function.Building upon in vitro studies that show ∼50 nM affinity forapoCaM interaction with channel CT, we utilized a medium-throughput flow cytometry–based fluorescence resonance en-ergy transfer (FRET) assay (46, 47) to demonstrate interactionbetween CaM and the wild-type (WT) NaV1.5 CT in live cells.We coexpressed Cerulean-tagged CaM as the FRET donor withVenus-tagged NaV1.5 CT as the FRET acceptor in HEK293 cells(Fig. 1D). Stochastic expression of the two interacting proteinsyielded variable FRET efficiencies (ED) that were quantifiedusing a flow cytometer from 5,000 to 20,000 individual cells. Asaturating binding curve was constructed by correlating ED withthe free acceptor concentration (Fig. 1D, black dots and fit). Thismaneuver yielded a relative binding affinity (Kd,EFF) indicative of

0 10000 20000Afree

0

0.3 ED0.2

0.1

WTS[1904]L

IQ/AA

CaM

EF1-2

IQ

NaV1.5 CT

C

NANaV1.5

CaM

EF1-2

IQBrSLQT3Mixed

h

0

1

V (mV)-120 -30V (mV)

1

-120 -300

h

C

0 mV

[Ca2+]( M)

1 nA

UV

-120

0.1

10

B

DIQEF

N C

NaV1.5 carboxy tail

VEN

VEN CT

CER

CER CaMWT

N CFRETpair

Fig. 1. Absence of dynamic Ca2+/CaM effects on WT NaV1.5 SSI. (A, Left)Structure of NaV1.5 transmembrane domain (6UZ3) (70) juxtaposed withthat of NaV1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linkedCT mutations highlighted in NaV1.5 CT–apoCaM structure (LQTS3, blue; BrS,magenta; mixed syndrome, purple). (B) Dynamic Ca2+-dependent changes inNaV1.5 SSI probed using Ca2+ photouncaging. Na currents specifying h∞ at∼100 nM (Left) and ∼4 μM Ca2+ step (Right). (C) Population data for NaV1.5SSI under low (black, Left) versus high (red, Right) intracellular Ca2+ reveal nodifferences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interactionwith various Venus-tagged NaV1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue).Each dot is FRET efficiency measured from a single cell. Solid line fits show1:1 binding isotherm.

2 of 12 | PNAS Kang et al.https://doi.org/10.1073/pnas.2025085118 Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent

arrhythmogenic phenotypes

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2


strong baseline binding between CaM and the WT NaV1.5 CT inlive cells (SI Appendix, Table S1). To dissect the functional con-sequences of apoCaM binding to NaV1.5, we utilized structure-guided and disease-linked mutations that diminish apoCaM in-teraction. We mutated the central IQ residues (I1908/Q1909)within the NaV1.5 IQ domain to double alanine (IQ/AA), whichwas shown to abolish apoCaM binding in in vitro studies (44, 48).Flow cytometry–based FRET analysis of the NaV1.5 IQ/AA mu-tant revealed diminished apoCaM binding (Fig. 1D, red dots andfit), with an ∼4,000-fold increase in Kd,EFF (SI Appendix, TableS1). We subsequently tested the channelopathic mutant S[1904]Lthat is linked to both BrS (13) and LQTS3 (9) based on its locationwithin the IQ–CaM C-lobe interface. Flow cytometric FRETanalysis between the S[1904]L CT and apoCaM demonstrated an∼44-fold weaker affinity compared to WT (Fig. 1D, blue dots andfit; SI Appendix, Table S1).

Disruption of apoCaM Binding Diminishes NaV1.5 Peak Open Probability.Having confirmed disruption of CaM binding by IQ/AA and S[1904]L mutations in live cells, we tested whether these mutationsalter NaV1.5 gating. We first probed SSI (h∞) properties withwhole-cell recordings. To discern apoCaM-dependent effects, weutilized high intracellular Ca2+ buffering in the pipette dialysate.A comparison of h∞ curves for WT channels versus IQ/AA and S[1904]L mutants revealed no appreciable shift in SSI (Fig. 2 A–Cand SI Appendix, Fig. S1J). Diminishing cytosolic CaM levels byoverexpressing a CaM chelator or enhancing cytosolic CaM byexogenous expression of recombinant CaM also did not alter theh∞ curves for WT, IQ/AA, and S[1904]L (Fig. 2 A–C and SIAppendix, Fig. S1J). By contrast, analysis of peak-current densitiessuggests CaM-dependent changes (SI Appendix, Fig. S1). Upondepletion of free CaM levels by overexpression of a CaM chelator,

the WT channels showed reduced peak-current density (SI Ap-pendix, Fig. S1 A and B). Similarly, both IQ/AA and S[1904]Lmutants with defective CaM binding also showed an approxi-mately threefold reduction in peak-current density compared toWT channels (SI Appendix, Fig. S1 A, D, and G). ExogeneousCaM overexpression with IQ/AA and S[1904]L mutants increasedthe peak-current density (SI Appendix, Fig. S1 F and I). Of note,we observed minimal differences in voltage dependence of activa-tion between the three channel constructs at different CaM levels(SI Appendix, Fig. S1K). These findings suggest that apoCaMbinding may be critical for baseline channel function. Alterations inpeak current could result from changes in surface membrane traf-ficking, unitary conductance, or peak open probability (PO/peak), anambiguity that may be readily resolved at the single-channel level.We undertook low-noise single-channel cell-attached record-

ings from HEK293 cells transfected with either NaV1.5 WT, IQ/AA, or S[1904]L mutant channels. Channel openings were eli-cited using step depolarizations from a holding potential of −120mV, ensuring maximal channel availability (Fig. 1 A–C). Thus,measured alterations in peak PO reflect changes in channelopenings from the resting state and not a shift in SSI. Exemplartraces in Fig. 2D show WT single-channel openings in responseto a step depolarization to −10 mV. The unitary current levelsat −10, −30, and −50 mV test voltages were estimated usingamplitude histograms yielding a single-channel conductance of∼18 pS (SI Appendix, Fig. S2A), consistent with previous singleNa channel recordings (49, 50). The ensemble average currentfrom ∼100 to 200 stochastic sweeps at each voltage was dividedby the unitary current and the number of channels in the patch toobtain a time-resolved open probability (PO) waveform (Fig. 2E),reflecting rapid channel activation and inactivation. Populationanalysis of WT PO/peak obtained at three different voltages and

-120

10 mV

100 ms

PO

0

0.5

NaV1.5 IQ/AA

I

H

G

0.5

0

PO/peak

V (mV)-100 -60 -20 20-80 -40 0 40

PO

0

0.5

NaV1.5 S[1904]L

L

K

J

PO/peak0.5

0

V (mV)-100 -60 -20 20-80 -40 0 40

-120 3 msNaV1.5 wildtype

D

0.5

0

PO/peakF

-10 mV

PO

0

0.5

E

V (mV)-100 -60 -20 20-80 -40 0 40

i = -1.5 pA

-130 0V (mV)0

1

h

-130 0V (mV)0

1 control+ CaM+ CaMchelator

-130 0V (mV)0

1

hh

NaV1.5 IQ/AA NaV1.5 S[1904]LNaV1.5 wildtype B CA

****

Fig. 2. Disruption of apoCaM preassociation diminishespeak PO of NaV1.5. (A) SSI (h∞) curve for WT NaV1.5measured under endogenous (black), low (red), and high(blue) CaM levels. Low CaM levels were attained byoverexpressing a CaM chelator. High CaM levels wereattained by overexpressing recombinant CaM. (Inset)Voltage protocol. (B and C) Format as in A but for NaV1.5IQ/AA (B) and S[1904]L (C) mutants. (D) Exemplar tracesshowing stochastic records of channel openings from aone-channel patch of NaV1.5 WT evoked in response to adepolarizing pulse to −10 mV. Solid line denotes zero-current baseline, and channel openings are downwarddeflections to the unitary current level (dashed line). (E)Ensemble average PO waveform from a single patch (147sweeps) reveals a PO/peak ∼0.5. (F) Population PO/peak val-ues are plotted as block dots and bars (mean± SEM) as afunction of the activating test pulse potential (n = 7patches, 1,768 sweeps/voltage). The black line is the singleBoltzmann fit for PO/peak–V relation with parameters:PO,max = 0.48; V1/2 = −36 mV, and slope factor (SF) = 9.5.(G–I) Format as inD–F but for NaV1.5 IQ/AA. (H) EnsemblePO average shown from a single patch with 120 sweeps.(I) Population data of NaV1.5 IQ/AA PO/peak–V relationshipis shown by pale red dots (mean ± SEM, n = 5, 1,088sweeps/voltage) and red line fit. Boltzmann fit parame-ters: PO,Max= 0.21; V1/2=−45 mV; SF= 9.5. Gray line is theWT fit reproduced from F. (J–L) Format as in D–F but forNaV1.5 S[1904]L. (K) Ensemble PO average obtained from254 sweeps in a single patch. (L) Population data forNaV1.5 S[1904]L PO/peak–V relationship is shown by pale reddots (mean ± SEM, n = 6, 1,365 sweeps/voltage) and redline fit. Boltzmann fit parameters for NaV1.5 S[1904]L:PO,Max= 0.26; V1/2 = −45 mV; SF = 9.5. Gray line is the WTfit reproduced from F. Statistical analysis: two-wayANOVA followed by post hoc Tukey’s multiple compari-sons test, **P < 0.01 with WT channels as reference.

Kang et al. PNAS | 3 of 12Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergentarrhythmogenic phenotypes


PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2
















across multiple cells and transfections revealed a maximal PO/peak of∼0.5 at −10 mV test pulse (Fig. 2F). By comparison, IQ/AA andS[1904]L mutant channels showed a reduction in channel openings,although rare sojourns to the open state appear qualitatively pro-longed (Fig. 2 G and J, respectively, quantified later; see Fig. 4).Single-channel conductance for both mutant channels were un-perturbed compared to WT (SI Appendix, Fig. S2 C and E).However, the PO/peak for both IQ/AA and S[1904]L mutantswere reduced by 2.5-fold as shown by ensemble average POwaveforms (Fig. 2 H and K) and the population data (Fig. 2 I andL). These results demonstrate that CT mutations disruptingapoCaM binding diminish PO/peak, an effect consistent with theloss-of-function BrS phenotype.

ApoCaM Binding Tunes Peak Open Probability of Mutant NaV1.5. Thereduction in NaV1.5 PO/peak by CT mutations hints that apoCaMpreassociation may be a critical determinant for basal activity.However, two ambiguities obfuscate the effect of CaM onNaV1.5 activity. First, the reduced PO/peak may not directly relateto weakened apoCaM preassociation; instead, it may result fromidiosyncratic structural alterations. Second, if weakened apoCaMbinding is responsible for reduced PO/peak, then it is unknownwhether CaM binding is obligatory for channel openings. Thelow residual activity of mutant channels may correspond topartial CaM occupancy of mutant channels.We reasoned that if the reduction in PO/peak for CT mutants

reflect a genuine loss of apoCaM binding, then overexpression ofCaM with CT mutant channels would by mass action repopulateCT mutant channels and thereby reverse functional defects inPO/peak. Indeed, previous studies have shown that CaM over-expression can restore CaM preassociation to mutant CaV1.3with diminished CaM-binding affinity (51). Single-channel re-cordings of both the IQ/AA and S[1904]L mutant in the presenceof high CaM revealed gating behavior qualitatively similar to WTchannels (IQ/AA, Fig. 3A; S[1904]L, Fig. 3E), with most sweepsfeaturing a single brief opening with fewer blank sweeps (quan-tified later, Fig. 4). Ensemble average PO waveforms showedincreased PO/peak for both IQ/AA (Fig. 3C, blue) and S[1904]L(Fig. 3G, blue) mutant channels, reversing the effects seen atendogenous CaM levels. Population PO/peak–V relationship in thepresence of CaM demonstrated statistically significant PO/peak in-crease compared to that measured under endogenous CaM con-ditions (Fig. 3 D and H, blue). By contrast, the single-channelconductance was unperturbed (SI Appendix, Fig. S2 D and F).The ability of heterologously expressed CaM to reverse deficits inPO/peak for CT mutants suggest that apoCaM interaction sufficesfor high basal channel activity.We next sought to determine whether residual channel activity

observed for IQ/AA and S[1904]L mutations correspond to partialCaM binding as a result of high endogenous CaM levels inHEK293 cells. If so, the depletion of ambient CaM concentrationby overexpression of a CaM chelator would result in further re-duction in PO/peak (52). Single-channel records of IQ/AA followingCaM depletion showed sparse but prolonged channel openingswith many blank sweeps (Fig. 3B; quantified later, Fig. 4). Analysisof the sweeps revealed a unitary conductance of 17 pS and a PO/

peak – V relationship ∼2.5-fold lower than that of WT (Fig. 3 C andD, red and SI Appendix, Fig. S2D). This relationship is nearlyidentical to the IQ/AA results obtained under endogenous levels ofCaM, suggesting that IQ/AA mutant channels lack appreciableCaM binding at baseline. We observed a similar trend with the S[1904]L mutation, whereby channels exhibit a reduced PO/peakupon CaM chelation much as with endogenous CaM concentration(Fig. 3 F–H, red). Consistent with these findings, reducing ambientfree CaM concentration also resulted in a modest (21.8%) re-duction in PO/peak for WT channels (SI Appendix, Fig. S3). Theseresults suggest that apoCaM preassociation is not obligatory forNaV openings; rather, it tunes baseline level of channel activity.

PO

0

0.5

V (mV)-100 -60 -20 20-80 -40 0 40

0.5

0

PO/peak

G

H

CaM chelator

E CaM

F

NaV1.5 S[1904]LNaV1.5 IQ/AA

3 ms

-10

-120 mV

A

0.5

C

PO

0

0.5

PO/peak

0

D

B CaM chelator

CaM

V (mV)-100 -60 -20 20-80 -40 0 40

******

***

**

Fig. 3. ApoCaM binding tunes maximal PO of mutant NaV1.5 with reducedCaM binding. (A and B) Exemplar stochastic single-channel recordings ofNaV1.5 IQ/AA under CaM-overexpressed (A) and CaM-depleted (B) condi-tions. (C) Ensemble PO average of NaV1.5 IQ/AA from a single patch underCaM-overexpressed (blue, 130 sweeps) and CaM-depleted (red, 276 sweeps)conditions. (D) Population NaV1.5 IQ/AA PO/peak–V relationship under CaM-overexpressed (blue dots and fit, n = 5, 539 sweeps/voltage) and CaM-depleted (red dots and fit, n = 7, 1,249 sweeps/voltage) conditions. Gray isthe NaV1.5 IQ/AA fit under endogenous CaM levels reproduced from Fig. 2I.All dots and bars are mean ± SEM. Boltzmann fit parameters for CaM-overexpressed condition (blue): PO,max = 0.45; V1/2 = −38 mV; SF = 9.5.Boltzmann fit parameters for CaM-depleted condition (red): PO,max = 0.23;V1/2 = −40 mV; SF = 9.5. Statistical analysis: two-way ANOVA followed bypost hoc Tukey’s multiple comparisons test, ***P < 0.001 comparing the CaMoverexpression with the CaM-depleted condition. (E and F) Format as in A–Dbut for NaV1.5 S[1904]L mutant. (G) Ensemble PO average of NaV1.5 S[1904]Lfrom a single patch under CaM-overexpressed (blue, 120 sweeps) and CaM-depleted (red, 218 sweeps) conditions. (H) Population NaV1.5 S[1904]L PO/

peak–V relationship under CaM-overexpressed (blue dots and fit, n = 11,2,496 sweeps/voltage) and CaM-depleted (red dots and fit, n = 8, 1,568sweeps/voltage) conditions. Gray is the NaV1.5 S[1904]L fit under endoge-nous CaM levels reproduced from Fig. 2L. Boltzmann fit parameters for CaM-overexpressed condition (blue): PO,Max = 0.5; V1/2 = −43 mV; SF = 9.5.Boltzmann fit parameters for CaM-depleted condition (red): PO,Max = 0.18;V1/2 = −45 mV; SF = 9.5. Statistical analysis: two-way ANOVA followed bypost hoc Tukey’s multiple comparisons test, **P < 0.01 and ***P < 0.001comparing the CaM overexpression with the CaM-depleted condition.



Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2






ApoCaM Interaction Switches State Dependence of NaV1.5 Inactivation.We sought to leverage information from single-channel recordingsto infer apoCaM-dependent elementary changes in NaV1.5 gatingfollowing the Aldrich–Corey–Stevens formalism. We analyzedone-channel patches to quantify the following: 1) FL(t), the firstlatency distribution describing the probability that the first open-ing will occur by time t [i.e., FL(t) = P(first opening ≤ t)]; 2) OD,the open-duration distribution that describes the total duration ofeach opening [i.e., OD(t) = P(duration of opening ≥ t)]; 3) NO, thenumber of openings in each sweep; and 4) POO (t + tj, tj), theconditional probability of observing a channel opening at time t+tj,if the channel were open at time tj. To dissect CaM-dependenteffects, we compared properties of IQ/AA mutant channels at ei-ther low or high levels of CaM. We focused on three aspects ofNaV1.5 gating in our analyses as conceptualized by the gating modeldrawn in Fig. 4I: 1): closed-state inactivation (kCI), 2) open-stateinactivation (kOI), and 3) burst-like behavior.We first examined close-state inactivation by analyzing FL dis-

tribution for NaV1.5 WT channels at −10 mV, which revealed asaturating exponential curve with a plateau value of FLmax ∼0.6.This indicates a ∼40% likelihood for the channel to inactivatewithout opening, that is, undergo closed-state inactivation (Fig. 4A).By comparison, the IQ/AA mutant channels at low CaM levelsshowed a decrease in the plateau value (FLmax ∼0.25) without achange in the time constant (Fig. 4B, red). This suggests that therate constant for closed to open transitions are unaltered; rather,the rate of transition from the closed to inactivated state is en-hanced. CaM overexpression reversed this change in FL for the IQ/AA mutant (Fig. 4B, blue), indicating that the change in FL dis-tribution results from diminished CaM preassociation. A similartrend was observed with test pulse potentials of −30 and −50 mV(SI Appendix, Fig. S4). The S[1904]L mutant also exhibited similarchanges in FL distribution at high versus low levels of CaM (SIAppendix, Fig. S5). In all, CaM-dependent alterations in FLmax areconsistent with apoCaM binding reducing the propensity for closed-state inactivation by decreasing kCI (Fig. 4I).We next scrutinized open-state inactivation with the OD dis-

tribution, which describes the time spent in the open state duringeach sojourn. For WT, the OD distribution was approximated bya single-exponential decay (Fig. 4C) with a time constant in-versely related to the sum of rate constants for the channelleaving the open state to the closed state (kOC; Fig. 4I) and to theinactivated state (kOI; Fig. 4I). The OD distribution of IQ/AAunder low CaM levels (Fig. 4D, red) was prolonged with a slowerexponential decay, reflecting long openings in exemplar records(Fig. 3B). CaM overexpression with the IQ/AA mutant reducedthe time constant for OD distribution, indicating briefer openings(Fig. 4D, blue) compared to low CaM levels. A similar trend wasobserved with −30 and −50 mV test pulse potentials (SI Appendix,Fig. S4) as well as in the S[1904]L mutant when the test pulsepotential was −10 mV (SI Appendix, Fig. S5). Taken together,these findings suggest that apoCaM binding increases the rate ofegress from the open state, which may result from either an in-crease in the rate of channel closure (kOC) or enhanced inacti-vation from the open state (kOI). To discern between thesepossibilities, we considered the distribution of the number ofopenings observed per each sweep (NO) for WT (Fig. 4E) and IQ/AA mutant channels (Fig. 4F). We reasoned that if apoCaMbinding only increased kOC, then channels would flicker betweenopen and closed states multiple times before inactivating. That is,an increase in kOC rate alone would cause a decrease in thelikelihood of observing exactly one channel opening provided thatthe channels opened at least once. However, this was not the case.The proportion of sweeps with exactly one opening when blanksweeps are excluded [i.e., P(NO = 1 | NO ≥ 1)] was similar betweenthe IQ/AA mutant under low (65%) and high CaM levels (68%)as well as WT channels (67%). Taken together, these resultssuggest that the prolonged OD seen in IQ/AA mutant channels at

low CaM levels reflects decreased kOI. At the macroscopic level,this change would result in a slowed current decay kinetics uponCaM depletion. Indeed, a single-exponential fit of whole-cellcurrent decay kinetics revealed prolonged decay kinetics at lowambient CaM levels as compared with CaM overexpression forWT NaV1.5 and both IQ/AA and S[1904]L mutant channels (SIAppendix, Fig. S6). In total, apoCaM binding likely enhances therate of NaV1.5 open-state inactivation.Lastly, we examined burst-like behavior by computing the

conditional open probability, POO(t), that depends on the follow-ing: 1) how long the channel stays open from the initial openingand 2) the tendency for the channel to reopen following closure.The WT NaV1.5 POO(t) relation followed an exponential decaythat was well approximated by the OD distribution (Fig. 4G), in-dicating that these channels favor complete inactivation after thefirst opening. By comparison, the POO(t) curve for the IQ/AAmutant at low CaM levels deviated from the OD distribution to asmall pedestal value (Fig. 4H, red). With CaM overexpressed, thischange was reversed for the IQ/AA mutant with the POO(t) curvebeing well approximated by the OD distribution (Fig. 4H, blue).The small but nonzero pedestal value for the POO(t) curve for theIQ/AAmutant at low CaM levels reflects a small fraction of sweepsexhibiting frequent channel reopenings or bursts (1.25% of sweepsfrom IQ/AA at low ambient CaM versus 0.3% at high CaM levels).These findings suggest that the disruption of CaM binding maylead to a small but pathophysiologically relevant persistent Nacurrent, a possibility that will be subsequently evaluated.In-depth analysis of the single-channel recordings suggests

three distinct functional effects related to apoCaM preassocia-tion on NaV1.5: 1) decreased rate of inactivation from the closedstate (kCI), 2) increased rate of inactivation from the open state(kOI), and 3) decreased tendency for channels to exhibit burst-like behavior. To further corroborate these findings, we imposeda Markov model depicted in Fig. 4I. We fit FL, OD, and POcurves for WT and IQ/AA mutants holding α, β, kCO, and kOCvalues constant across all conditions while allowing kCI to in-crease and kOI to decrease as a consequence of loss of apoCaMbinding (Fig. 4I, bar plot). This minimalistic model reasonablyapproximated the PO, FL, and OD curves across all constructs atthree test voltages (solid line fits, Fig. 4 A–D, G, and H and SIAppendix, Figs. S4 and S5). Taken together, apoCaM-dependentchanges in PO/peak reflect a switch in the state dependence ofchannel inactivation, with the loss of apoCaM binding biasingpreferential closed-state inactivation.

ApoCaM Interaction with NaV1.5 Tunes Persistent Current. Analysisof NaV1.5 singe-channel recordings show that the loss of apoCaMpreassociation results in a pedestal level of the POO distribution,hinting at increased channel reopenings. Indeed, previous studiessuggest that the loss of apoCaM preassociation increases a per-sistent Na current, an important arrhythmogenic substrate (44).To elucidate CaM-dependent changes in a persistent Na current,we undertook cell-attached multichannel recordings from eitherWT or mutant NaV1.5. Unlike whole-cell approaches that may besusceptible to a nonspecific membrane leak, late Na channelopenings can be reliably detected with the multichannel approach,as the channel unitary conductance is sufficiently large to discernsingle openings from instrument noise (53).An exemplar stochastic record of WT NaV1.5 evoked in re-

sponse to a 300-ms step depolarization to −30 mV shows rapidchannel activation as indicted by stacked openings followed byrapid inactivation with no openings in the late phase, defined asfollowing 50 ms of depolarization (Fig. 5 A, Top, gray shadedarea and inset). For each patch, we averaged 50 to 100 stochastictraces to obtain an ensemble average current, which is subsequentlynormalized by the peak value to obtain the normalized PO waveform(Fig. 5A,Bottom and SI Appendix, Fig. S7). As ametric for the persistent



PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2













current, we quantified the average value of the normalized PO curvein the late phase for each patch (Rpersist = < PO > 50 ≤ t ≤ 300/PO/peak).This measurement is analogous to whole-cell measurement of latecurrents quantified as a ratio of the steady-state Na current nor-malized by the peak. WT channels exhibited a minimal persistentcurrent as evident from population Rpersist values (Fig. 5J), indi-cating near complete channel inactivation. We subsequently pro-bed whether the depletion of CaM binding to WT channels tunethe persistent current by overexpressing a CaM chelator (Fig. 5B).This maneuver yielded channel openings in the late phase (Fig.5B) and a statistically significant increase in Rpersist values (Fig. 5J).By contrast, CaM overexpression resulted in no change in Rpersistcompared to control conditions (Fig. 5 C and J). These findingssuggest that a loss of apoCaM interaction with NaV1.5 WT in-creases the persistent Na current.Thus informed, we measured the persistent current for the IQ/

AA mutant NaV1.5 whose CaM binding ability is strongly di-minished. Even at endogenous levels of CaM, the IQ/AA mutantexhibited a 10-fold increase in Rpersist (Fig. 5 D and J). Coex-pression of the CaM chelator did not further increase the latecurrent (Fig. 5 E and J), consistent with the absence of baselineCaM binding to these channels. Overexpression of CaM with theIQ/AA mutant, however, resulted in a partial inhibition of persis-tent channel activity (Fig. 5 F and J), presumably reflecting a partialrestoration of CaM preassociation. To determine whether the lossof CaM preassociation due to channelopathic mutations can causeincreased persistent activity, we probed the S[1904]L mutant. Withendogenous levels of CaM, we observed an increase in persistentchannel openings compared to WT (Fig. 5 G and J), reflectingweakened CaM binding. Decreasing ambient CaM levels furtherincreased Rpersist values to NaV1.5 IQ/AA levels (Fig. 5 H and J),while CaM overexpression decreased the persistent current to nearWT levels (Fig. 5 I and J). In-depth analysis suggests that changes inRpersist pertain to altered CaM occupancy of channels (SI Appendix,Text). Taken together, these findings demonstrate that CaM bind-ing to NaV1.5 tunes the likelihood of persistent channel openings.If both PO/peak and Rpersist modulation relies on the preassoci-

ation of a single apoCaM to the channel CT, then we would expectthe two entities to follow a linear relation, implicitly representingvaried apoCaM binding. As Rpersist values were obtained frommultichannel patches, we computed a corresponding PO/peak for allpatches by estimating the number of channels in each patch usingmean-variance analysis. Plotting Rpersist versus PO/peak demon-strated a linear relationship confirming that the preassociation ofthe very same CaM elicits both effects (Fig. 5K). Importantly,multiple CT channelopathic mutations variably weaken CaMbinding. We expect that these mutations will result in a variableincrease in the level of persistent Na current with up to an order ofmagnitude change.

Ca2+/CaM Interaction with NaV1.5 Tunes Persistent Current. NaV CTchannelopathies have been shown to differentially impactapoCaM versus Ca2+/CaM interaction. One possibility is thatchanges in intracellular Ca2+, as expected during cardiac systole,could impact the dynamic equilibrium of CaM association withNaV1.5. This change in the CaM-binding status of NaV1.5 could inturn dynamically tune persistent channel activity and thereby af-fect AP repolarization. To dissect this possibility, we employedflow cytometric FRET two-hybrid assays to determine Ca2+-de-pendent changes in CaM binding to the NaV1.5 WT, IQ/AA, andS[1904]L CT (Fig. 6A and SI Appendix, Table S1). Compared to itsbinding affinity in the apo-configuration, Ca2+-bound CaM features

0 1.5E-30 10 0 50 0 1ms-1 ms-1 kCO ms-1 kOC ms-1 kCI ms-1

0 10kOI ms-1

0 4

WT

IQ/AA

C1 O3kCO

I4 I5

C2 kOCkOIkCI

CaM C1 O3

I4 I5

C2

I

+ CaM- CaM

P(Topen>t)

0

1

0 1 2 3 4 5 6t (ms)

0 1 2 3 4 5 60

1

OpenDuration

FirstLatency

0 1 2 3 4 5 6t (ms)

0

1F(t)

AP(To pen>t)

0 1 2 3 4 50

1

6

C

t (ms)

0

1

0 1 2 3 4 5 6

POO(t)

G H

0

1

0 1 2 3 4 5 6t (ms)

POO(t)

Pedestal

t (ms)

NaV1.5 wildtype

F(t)

B

D

NaV1.5 IQ/AA

+ CaM- CaM

CaM

CaM

F

0

1

0 1 2 3 4 5

P

0

1

P

0 1 2 3 4 5# of openings / trace # of openings / trace

E

t (ms)

***

Fig. 4. Elementary mechanisms underlying CaM regulation of NaV1.5. (Aand B) FL distributions for NaV1.5 WT (gray shaded area) and NaV1.5 IQ/AAmutant under CaM-overexpressed (pale blue shaded area) and CaM-depleted (rose shaded area) conditions. FL denotes the probability thatthe first opening occurred at time < t. Solid lines are fits of FL distributionsgenerated by the model shown in Fig. 4I. The difference in FL distributionsfor NaV1.5 IQ/AA at low versus high CaM levels are statistically significant(P < 0.001, Kolmogorov–Smirnov [KS] test). (C and D) Histograms of ODdistribution which correspond to the time spent in the open state for WT(gray shaded area, C) and IQ/AA under low (rose shaded area, D) and high(pale blue shaded area, D) CaM conditions. Solid lines are fits of OD distri-butions generated by the model shown in Fig. 4I. The OD distribution of IQ/AA is significantly prolonged under low CaM levels compared to CaMoverexpression (D, P < 0.001, KS test). (E and F) Distribution of the number ofopenings per sweep for WT (black) and IQ/AA mutant channels at low (red)and high (blue) levels of ambient CaM. ***P < 0.001, test of proportion forcomparing fraction of blank sweeps. (G and H) Conditional open probability(POO(t)) for WT (gray shaded area) and IQ/AA at low (rose shaded area) orhigh (pale blue shaded area) CaM levels. Solid lines are reproduction of thefits of OD distributions in C and D. For NaV1.5 IQ/AA, POO(t), distributions atlow versus high CaM are statistically different (P < 0.001, KS test). (I) Sum-mary of the effect of CaM on NaV1.5 gating. Absent CaM, channels prefer-entially inactivate from the closed state. Upon binding CaM, inactivation

proceeds preferentially from the open state (α: rate of transition from theclosed to open state, β: rate of transition from the open to closed state, kOI:rate of inactivation from the open state, kCI: rate of inactivation from theclosed state, kOC: rate of channel closure, and kCO: rate of channel opening).



Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2





only a modest change in affinity for the WT NaV1.5 CT domain(Fig. 6B and SI Appendix, Table S1). By contrast, Ca2+/CaM ex-hibits an ∼35-fold increase in binding affinity over that of apoCaMfor the IQ/AA mutant (Fig. 6B and SI Appendix, Table S1). The CTdomain of the S[1904]L mutant demonstrated similar binding af-finities to both apo- and Ca2+/CaM, with both being reducedcompared to WT (Fig. 6B and SI Appendix, Table S1). These dis-tinct patterns of apo- versus Ca2+/CaM binding to the mutant IQdomains are consistent with prior studies (24, 27, 38, 44).To probe dynamic Ca2+-dependent changes in the late Na+

current, we coexpressed NaV1.5 with CaV2.1 channels and un-dertook cell-attached multichannel recordings to determinewhether Ca2+ spillover from neighboring Ca2+ channels perturblate NaV1.5 openings (32). We employed the voltage protocolshown in Fig. 6C, which enables persistent Na current quantifi-cation and a clear delineation between NaV1.5 versus CaV2.1activity (SI Appendix, Fig. S8). As CaV2.1 requires strong depo-larization to activate, NaV openings alone could be measured bya modest depolarizing “prepulse” to −35 mV (Fig. 6C, pulse i).The same voltage protocol applied to cells transfected withCaV2.1 alone exhibited no channel activity at −35 mV (SI Ap-pendix, Fig. S8 A and B). We then applied an “intervening pulse”to +15 mV to activate CaV2.1, permitting local Ca2+ influx andspillover (Fig. 6C, pulse ii). Lastly, a “postpulse” to −35 mVenabled measurement of persistent NaV1.5 openings followinglocal Ca2+ elevation (Fig. 6C, pulse iii). We expressed exogenousCaM to ensure that sufficient CaM is available to act on NaV1.5as CaV2.1 also binds CaM with a high affinity.Fig. 6 C and D displays an exemplar trace of the Na persistent

current measured from WT channels with this protocol alongwith the normalized PO waveform from the ensemble average.The WT NaV1.5 persistent current following Ca2+ entry exhibitedminimal change compared to that during the prepulse (Fig. 6 C, D,

and I). By contrast, the IQ/AA mutant revealed an ∼10-fold re-duction in persistent channel openings following Ca2+ influx viaCaV2.1 (Fig. 6 E, F, and I). We further probed whether the per-sistent current reduction in IQ/AA was Ca2+ dependent by re-peating the experiment in cells transfected with IQ/AA alone.Absent Ca2+ entry through CaV2.1, we observed no difference inpersistent NaV openings of the IQ/AA mutant following the in-tervening pulse (SI Appendix, Fig. S8 C–E). This suggests that thereduction in the persistent current of IQ/AA reflects a genuineCa2+-dependent effect. The S[1904]L mutant exhibited mini-mal change in the persistent current following Ca2+ spillover(Fig. 6 G–I). Mechanistically, Ca2+-dependent changes in lateNaV openings correlate with Ca2+-dependent changes in CaMbinding to NaV1.5 CT. Specifically, both WT and S[1904]L mu-tants exhibited a minimal change in CaM binding, whether in apo-or Ca2+-bound configuration. Functionally, late Na channelopenings of both channels were insensitive to dynamic changes inCa2+. By comparison, the IQ/AA mutant exhibits a 35-fold in-crease in CaM affinity in the presence of Ca2+. Functionally, thischange results in a reduction in late channel openings followinglocal Ca2+ spillover, presumably reflecting CaM recruitment tothe channel. These findings suggest that dynamic Ca2+ fluctua-tions may tune late NaV openings; however, manifestation of thiseffect and its magnitude may be idiosyncratic depending on theprecise mutation and the overall changes in apo- versus Ca2+/CaM binding.

Late NaV1.5 Openings Are Prolonged Similar to Inactivation-DeficientChannels. Given the increase in the persistent Na current resultingfrom the loss of CaM binding and its potential importance to cardiacarrhythmogenesis, we further scrutinized biophysical mechanismsunderlying the CaM-dependent late Na current. We calculated theconditional OD distributions for channel openings that occur

A B C

D E F

G H I

J K

Fig. 5. ApoCaM interaction with NaV1.5 tunes like-lihood for persistent channel openings. (A, Top)Representative multichannel record from NaV1.5 WTshows rapid activation and inactivation followed byrare openings in the late phase following 50 ms ofdepolarization (gray shaded region). (Inset) Enlargedlate phase to better visualize NaV1.5 openings.(Bottom) Normalized ensemble average open prob-ability (n = 16, 875 sweeps). (Inset) Enlarged nor-malized open probability in the late phase. (B and C)Format as in A Top but for NaV1.5 WT channelsrecorded under CaM-depleted (B) and CaM-overexpressed (C) conditions. (D–I) Format as in A–Cbut for NaV1.5 IQ/AA (D–F) and S[1904]L (G–I) mu-tants. (J) Quantification and population data forpersistent channel openings for NaV1.5 WT, IQ/AA,and S[1904]L under different CaM concentrations(A–I). For each condition, a normalized ensembleopen probability PO is calculated by averaging manysweeps (SI Appendix, Fig. S7). Rpersist is the averageopen probability PO in the late phase (gray shadedregions in A–I) normalized by the peak PO. Each barand error, mean ± SEM. Statistical analysis: one-wayANOVA followed by Tukey multiple comparisonstest. ***P < 0.001, **P < 0.01, *P < 0.05 compared toWT at endogenous CaM levels; ##P < 0.01 versus IQ/AA at endogenous CaM level; and ††P < 0.01 whencompared to S1904L at endogenous CaM levels. (K)Correlation of Rpersist versus PO/peak under differentCaM concentrations shows a linear relationship.



PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2











within the late phase (greater than 50 ms after depolarization) forWT, IQ/AA, and S[1904]L channels (Fig. 7 A and C–F, pale blue)and compared to those in the early phase (20 ms of depolariza-tion) (SI Appendix, Fig. S4 E and F). As late channel propertieswere determined from multichannel patches, we limited ouranalysis to openings that occur to the one-channel level. Theconditional OD distributions obtained for WT, IQ/AA, and S[1904]L all appeared biexponential and prolonged compared toearly openings (Fig. 7 A and C–F; blue shaded region and fit forlate openings, versus gray solid line for early openings reproducedfrom SI Appendix, Fig. S4 E and F). The shared biexponential ODdecay suggests that late openings may arise from two distinct openstates: one open state from which inactivation stems normally andanother from which inactivation is diminished corresponding tothe persistent mode. To examine whether the slow component ofthe conditional OD distributions may reflect an “inactivation-deficient” open state, we undertook single-channel recordings ofthe mutant NaV1.5 with the phenylalanine residue of the IFMmotif substituted with a glutamine residue (F1486Q; IFM/IQM).Consistent with previous studies showing that the IQM mutant isinactivation deficient (54, 55), this maneuver resulted in “burst-like” openings (SI Appendix, Fig. S9). The OD distribution of theIFM/IQM mutant exhibited prolonged decay that matched thetime constant of the slow component of the conditional late-phaseOD distribution inWT channels (Fig. 7 A and B, red fit). Thus, theslow component of the biexponential conditional OD distributionfor late channel openings may correspond to an equivalent of aninactivation-deficient open state. The same pattern of fast andslow components time constant matching was also observed in theconditional OD distributions for the IQ/AA and S[1904]L chan-nels under high and low CaM levels (Fig. 7 C–F). In all cases, thelate-phase conditionalOD distributions were well approximated asthe sum of two exponentials (dark blue fit; Fig. 7 A and C–F): the

first component with a time constant matching theOD distributiondetermined for each channel variant in the early phase (SI Ap-pendix, Figs. S4F and S5E) and the second component having atime constant that matched the OD distribution for IFM/IQMmutant (Fig. 7B). Importantly, the IQ/AA and S[1904]L mutantsat low CaM levels featured a higher fraction of the slow component(∼0.5) to WT (∼0.25) (Fig. 7 A, C, and E intercept of red fits), whileCaM overexpression reduced the magnitude of the slow componentin the mutant channels (intercept of red fits in Fig. 7C versusFig. 7D and Fig. 7E versus Fig. 7F). These findings are consistentwith the loss of CaM preassociation decreasing the channel pro-pensity to enter an “inactivation-deficient” open state. Taken to-gether, these results suggest that CaM tunes NaV1.5 occupancy ofan alternate open state akin to inactivation-deficit channels, therebymodulating the propensity for the persistent openings.

In Silico Modeling of CaM Regulation of NaV1.5 on Cardiac AP.Mechanistic analysis suggests that the disruption of apoCaMbinding to NaV1.5 reduces the peak current and increases thepersistent current. To discern the potential consequences ofthese changes, we modified the previously established ToR-ORdhuman ventricular action potential model to simulate the effectof reduced NaV1.5 apoCaM binding on cardiac AP morphology(56). We parameterized both the peak Na current amplitude andthe persistent current amplitude as functions of the fraction ofNaV1.5 bound to CaM (fb,CaM), while maintaining the empiricalrelationship between Rpersist and PO/peak as in Fig. 5K. Simula-tions performed at a low heart rate revealed that a reduction inCaM binding resulted in a nonlinear prolongation of both theepicardial and the endocardial AP duration (APD). A maximal1.8-fold increase in epicardial APD90 was observed with fb,CaM∼30% (SI Appendix, Fig. S10 A–C). This nonlinearly occurs as areduction in fb,CaM increases Rpersist but also decreases PO/peak,

0

0.3

0

ED

10000 20000Afree

A B

Ca2+

WT IQ/AA S1904L

Ka,EF

F(A

free-1 )

10-2

10-7

1000

4%

300

PO

t (ms)

0 100 300t (ms)

H

Na V1.5S[1904]L

G

1000

4%

300

PO

0 100 300

F

t (ms)

Na V1.5IQ/AA

E

1000

4%

300

0 100 300

25%

D

PO

PO

t (ms)

t (ms)

-120-35 mV +15

100 ms

Na V1.5WT

C

1pA

Ca2+

WT IQ/AA S1904L

0.01

0.1

1

10

100 PO/latePO/peak(%)

I10-3

10-4

10-5

10-6

(i)(ii)

(iii)

(i) (ii)

(iii)

(i)(ii)

(iii)

**

Fig. 6. Dynamic Ca2+ fluctuations tune persistentNaV channel openings. (A) FRET two-hybrid analysisprobes the interaction of Venus-tagged NaV1.5 CTwith Cerulean-tagged CaM at high Ca2+ levels. Blackdots and fit, WT; red dots and fit, IQ/AA mutant; bluedots and fit, S[1904]L mutant. (B) Bar graph summarycompares Ka,EFF = 1/Kd,EFF for Ca2+ versus apoCaMbinding to WT NaV1.5 as well as IQ/AA and S[1904]Lmutant. (C) Exemplar multichannel stochastic recordfrom cell-attached recordings of NaV1.5 WT coex-pressed with CaV2.1. NaV1.5 openings are evokedusing the −35 mV voltage pulse. Ensuing +15 mVvoltage pulse elicits Ca2+ channel openings (roseshaded area). A subsequent pulse to −35 mV is usedto identify Ca2+-dependent changes in persistent NaVchannel openings. (D) Normalized ensemble averagePO waveform is computed from 293 sweeps (sixpatches). A comparison of the PO waveform before(black) and after (red) Ca2+ pulse reveals minimaldifferences in the late Na current. (E–H) Exemplarmultichannel recordings and ensemble average PO

waveforms of NaV1.5 IQ/AA (E and F) or S[1904]L (Gand H) coexpressed with CaV2.1. Format same as Cand D. Data obtained from 342 sweeps (sevenpatches) for NaV1.5 IQ/AA and 264 sweeps (sevenpatches). (I) Paired dot plot summarizes changes inRpersist before (black dot, gray bar) and after (red dot,rose bar) Ca2+ influx. Only the IQ/AA mutant showeda consistent reduction in the late current followingCa2+ entry (**P < 0.01 by paired t test).



Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2









causing nonlinear changes in the net late Na+ conductance. Theincrease in APD is consistent with the LQTS3 phenotype ob-served with CT channelopathies.By comparison, the mechanism of BrS is thought to be a pre-

mature all-or-none repolarization of the epicardial but not endo-cardial AP, leading to dispersion of repolarization across theventricular wall and an increased susceptibility for reentry (10, 11).Although diminished apoCaM binding reduced the peak Na cur-rent in our simulations, the concomitant enhancement in thepersistent Na current provided a sufficient depolarizing influx toprevent premature AP repolarization. Thus, additional factorsthat tune the persistent Na current may be relevant for BrSpathogenesis of NaV CT channelopathies. One possibility is fi-broblast growth factor (FGF) homologous factors (FGF12B/13),which interact with NaV1.5 in cardiomyocytes (57) and inhibit thepersistent current for CT mutations (39, 53). FGF association withNaV1.5 CT mutants lacking apoCaM may lead to a reduction inpeak Na current without a concurrent increase in persistent Nacurrent (39). Simulations that account for the FGF effect dem-onstrate a loss of the AP dome in the epicardial but not endo-cardial AP in a heart rate–dependent manner (SI Appendix, Fig.S10 D–F). This behavior is consistent with a BrS phenotype. Al-together, these simulations indicate that the disruption of apoCaMbinding can lead to both LQTS3 or BrS phenotypes, with the latterpotentially requiring additional factors such as FGF12/13.

DiscussionCaM regulation of NaV channels is multifaceted and isoformspecific with complex underlying molecular mechanisms (14, 15,28, 58). This study provides an in-depth single-channel analysisof CaM regulation of cardiac NaV1.5. Having confirmed the ab-sence of dynamic Ca2+-dependent regulation on SSI, we focused onelucidating effects of apoCaM on NaV1.5. Disruption of CaMpreassociation via IQ/AA and mixed-syndrome S[1904]L mutationsevoked two disparate functional effects: 1) an up to threefold re-duction in peak open probability (PO/peak) and 2) an up to 10-foldincrease in the likelihood for late or persistent Na channel openings(Rpersist). For both mutants, CaM overexpression reverses changesin both PO/peak and Rpersist by repopulating channels with CaM bymass action. The correlation of PO/peak versus Rpersist revealed alinear relationship suggesting that the binding of a single apoCaMto the channel CT mediates both functional effects. Furthermore,computer simulations suggest that these biophysical changes alterAP morphology consistent with either LQT3 or BrS phenotypes.These findings lend long-sought mechanistic insights into CaMregulation and bear important implications for arrhythmogenicmechanisms underlying NaV1.5 channelopathies.Over the past two decades, CaM regulation of NaV channels

has been subject to intense scrutiny with considerable effort devotedto identifying Ca2+-dependent effects of NaV1.5 gating (14, 15, 28,58). Both functional manifestation and underlying mechanisms ofCa2+ regulation has remained controversial. Initial studies sug-gested that an EF hand in the NaV1.5 CT directly coordinates Ca2+

to alter SSI (59–61); however, subsequent structures excluded directCa2+ binding (30). Instead, CaM was purported to mediate Ca2+

effects (35). Multiple CaM-binding sites have been identified withinthe channel cytosolic domains including 1) an IQ domain in thechannel CT that binds both apoCaM and Ca2+/CaM (24–26, 28, 29,32), 2) a segment downstream of the IQ motif that interacts withCa2+/CaM (26, 29), 3) two Ca2+/CaM binding regions within theIII-IV linker (35, 37), and 4) the channel N terminus (36). Thefunctional importance of CaM binding to distinct sites has beendebated and typically involves variable Ca2+-induced shifts in SSI.Our results here demonstrate the absence of acute Ca2+ regulationof NaV1.5 SSI. One possibility is that the kinetics of Ca2+-inducedshifts in SSI are slow (>1 s) and only present during pathophysio-logical conditions. It is possible that posttranslational changes couldalso accelerate Ca2+/CaM effects (62).

Beyond dynamic Ca2+ effects, apoCaM fulfills a structuralrole as a de facto subunit responsible for conformational changes(44). ApoCaM binding has been shown to tune baseline functionof multiple ion channel families including CaV (58, 63), KV7 (64,65), and ryanodine receptors (66, 67). Previous studies have shownthat the disruption of CaM binding to NaV1.5 increases the per-sistent current (44). Our work extends this finding to the single-channel level and demonstrates that apoCaM binding also in-creases channel peak open probability. Interestingly, our resultsfurther show that either Ca2+/CaM or apoCaM binding to NaV1.5CT suppresses persistent current. This phenomenon may havecomplex and mutation-specific pathophysiological consequences,as disease mutations with distinct apoCaM- versus Ca2+/CaM-binding affinities may yield a variable late current (27, 68). Ca2+

regulation of the late Na current may also be relevant for muta-tions outside of the CaM-binding segment (68, 69). One limitationof the present study is that we focused on mutations in the channelCT to identify the putative effects of CaM. It is possible that CaM-dependent changes in PO/peak and Rpersist may partially reflectCaM association with additional CaM-interacting domains asnoted above. In particular, when considered in isolation, the IQ/AA mutation strongly disrupts apoCaM binding, yet CaM over-expression sufficed to reverse these changes to a large degree. It ispossible that in the context of the holo channel, additional CaM-binding sites in the N terminus (36) and in the III-IV loop (35, 37)may synergistically augment both apo- and Ca2+/CaM interaction.Mechanistically, CaM binding triggers a switch in state depen-

dence of channel inactivation. With apoCaM bound, channelspreferentially inactivate from the open state (OSi). Channels de-void of CaM exhibit enhanced closed-state inactivation (CSi) thatresults in an increased failure of channel openings and an increasedpropensity for late channel openings. Interestingly, the kinetics oflate NaV1.5 openings match channels with a defective inactivationgate. Recent cryogenic electron microscopy (cryo-EM) and crys-tallographic structures hint at potential changes underlying theCaM-dependent switch in channel gating (41, 42, 70). A compar-ison of the NaVPaS structure with that of mammalian NaV1.5shows a translocation of the III-IV linker from the proximal CT toa cleft adjoining the Domain IV (DIV) S6 gate (41, 42, 70). In vitroanalysis also confirms a weak interaction between the proximal CTand the III-IV linker (38, 43, 71), with alterations at this interfacemodifying the late Na current (39, 40). Further cryo-EM studies ofa hybrid NaV1.7-NaVPaS channel proposed a two-switch mecha-nism for NaV fast inactivation (42). At the resting state, the DIVgating charges interact with the CT (switch 1) to promote III-IVlinker/CT interactions (switch 2) (Fig. 7I). Upon membrane depo-larization, DIV gating charges move away from the CT and releaseswitch 1. This destabilizes switch 2, weakening III-IV linker associ-ation with the CT, and frees the IFMmotif to inactivate the channel.One possibility is that CaM regulation allosterically modifies the two-switch inactivation mechanism (Fig. 7I) by stabilizing switch 1 (DIV/CT) and destabilizing switch 2 (III-IV linker/CT). CaM stabilizationof switch 1 may prevent CSi by slowing DIV activation, while thedestabilization of switch 2 may enable OSi by promoting III-IVlinker dissociation from the CT following the release of switch 1.This study provides insights into potential pathogenic mecha-

nisms underlying arrhythmogenic NaV1.5 CT channelopathies.Clinically, NaV1.5 channelopathies within the CaM-binding in-terface of the channel CT exhibits variable penetrance with somemutations linked to either BrS, LQTS3, or both (6, 7, 9–11, 13).Our findings highlight the multifaceted and seemingly paradoxicalmodulation of cardiac NaV channels by CaM. Altogether, ourresults provide an in-depth molecular framework for divergentarrhythmogenic mechanisms and furnish possible explanations forhow certain channel CT mutations may yield mixed-syndromephenotypes.



PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2





Materials and MethodsMolecular Biology. The human NaV1.5 channel corresponds to clone M77235.1(GenBank). The CT domain of NaV1.5 was PCR amplified and subcloned into aZero Blunt TOPO II (Invitrogen) vector. To construct IQ/AA and S[1904]L mu-tations, we performed overlap extension PCR and ligated the mutated PCRproduct into the NaV1.5/pGW1 vector using KpnI and XbaI restriction sites. Forexperiments involving the CaM chelator, we utilized the Myosin Va IQ do-mains 3 to 6 attached to a Venus fluorophore (Ven-MyosinVa 4×IQ) in previousstudies (52). Venus-tagged NaV1.5 CT and Cerulean-tagged CaM were con-structed using the following strategy: We PCR amplified the CT of NaV1.5 withthe initial residue I[1771] and ligated to the 3′ end of a Venus pcDNA3 plasmidfollowing restriction digest using NotI and XbaI. Similarly, we PCR amplifiedCaM and ligated into the 3′ end of Cerulean pcDNA3 after restriction digestusing NotI and XbaI.

Cell Culture and Transfection. HEK293 cells were cultured on glass coverslips in60mm dishes and transfected using the Ca2+ phosphate method as previouslydescribed. For whole-cell patch clamp and cell-attached multichannel ex-periments, we cotransfected 4 to 8 μg complementary DNA (cDNA) encodingthe desired channel variant with 4 μg Yellow Fluorescent Protein (YFP) and1 μg simian virus 40 T antigen. Electrophysiology recordings were performedat room temperature 1 to 2 d following transfection.

For single-channel recordings, 1 μg NaV1.5 channel variant was trans-fected in 100 mm dishes using the Ca2+ phosphate method, and recordingswere performed at room temperature ∼12 h after transfection. For experi-ments manipulating ambient CaM levels, either 10 to 20 μg Ven-MyosinVa4×IQ DNA or 20 μg CaM DNA were used in CaM-depleted and CaM-overexpressed conditions, respectively.

Ca2+ Uncaging and Fluorescence Measurements. Ca2+ uncaging experiments(32) were performed using a Nikon TE2000 inverted microscope with a PlanFluor Apo 403 oil objective. Ca2+ was uncaged by ∼1.5 ms duration UV flashes(Cairn UV photolysis system). Flashes were driven by discharge of a 4,000 mFcapacitor bank charged to 200 to 300 V. Photomultiplier tubes were shutteredduring the UV pulse to prevent photo damage. For Ca2+ imaging, Fluo-4FF andAlexa 568 dyes (in fixed ratios) were dialyzed into cells and imaged with Argonlaser excitation (514 nm). The autofluorescence of each cell was obtainedbefore pipet dialysis. Single-cell fluorescence emission was isolated by a field-stop aperture. Dual-color fluorescence emission was obtained with a 545DCLPdichroic mirror paired with a 545/40 bandpass filter for Fluo-4FF and a 580longpass (LP) filter for Alexa 568. Uncaging was conducted after ∼2 min di-alysis. Steady-state [Ca2+] is measured 150 ms following UV pulse.

Whole-Cell Recordings. Whole-cell recordings were obtained at room tem-perature with an Axopatch 200B Amplifier (Axon Instruments). Electrodeswere made of borosilicate glass (World Precision Instruments, MTW 150-F4),yielding pipets of 1 to 2 MΩ resistance, which was compensated by >70%.Pipets were fabricated with a horizontal micropipette puller (model P-97,Sutter Instruments) and fire polished with a microforge (Narishige). Dataacquisition utilized an ITC-18 (InstruTech) data acquisition unit controlled bycustom MATLAB software (MathWorks). Currents were low-pass filtered at2 kHz before digitization at several times that frequency. P/8 leak subtractionwas used. Cells were maintained at a holding potential of −120 mV. The pipetsolution contained (in millimolars): CsMeSO3, 114; CsCl, 5; MgCl2, 1; MgATP, 4;HEPES (pH 7.4), 10; and BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), 10; at 290 mOsm adjusted with glucose. The bath solutioncontained (in millimolars): TEA-MeSO3, 20; HEPES (pH 7.4), 10; NaCl, 140; CaCl2,1.0; at 300 mOsm adjusted with TEA-MeSO3.

Single- and Multichannel Recordings. Cell-attached patch clamp experimentswere performed on HEK293 cells. The pipet solution contained the following(in millimolars): TeA-MeSO3, 30; NaCl, 140; CaCl2, 0.5; and Hepes, 10 (pH 7.4).The bath solution used for on-cell recordings of Na channels contained thefollowing (in millimolars): K-glutamate, 132; KCl, 5; NaCl, 5; MgCl2, 3; EGTA(ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), 2; glu-cose, 6; and Hepes, 10 (pH adjusted to 7.4). For experiments involving Ca2+

and Na channels, the pipet solution contained the following (in millimolars):TeA-MeSO3, 30; NaCl, 110; CaCl2, 10; and Hepes, 10 (pH 7.4). Data wereacquired at room temperature using the integrating mode of an Axopatch200A Amplifier (Axon Instruments). Patch pipettes (5–15 MΩ for single and3–8 MΩ for multichannel) were pulled from ultra-thick walled borosilicateglass (BF200-116–10; Sutter Instruments) using a horizontal puller (P-97,Sutter Instruments), fire polished using a microforge (Narishige), and coatedwith Sylgard (Dow Corning). Data acquisition used an ITC-18 16-bit low-noise

NaV1.5 IFM/IQM

0 8

0

1

P(TO>t)

t (ms)

C

NaV1.5 WT

early openingslate openings

0 8

0

1P(TO>t)

t (ms)

D

0 80

1


t (ms)

P(TO>t)

0 80

1


t (ms)

P(TO>t)

Na V1.5S[1904]L

0 80

1

t (ms)


P( TO>t)

+CaM+CaM chelator

0 80

1

t (ms)


Na V1.5IQ/AA

P(TO>t)

A

E F+CaM+CaM chelator

B

+

closed-inactivated

CaM

unbound

++

persistent mode

++

destabilizeswitch 1

stabilizeswitch 2

+

open++

open-inactivatedclosed

CaM

bound

+

+

V V

G

+

D4

+

switch 2switch 1

inactivation deficient

Fig. 7. In-depth analysis suggests a distinct open state associated with lateNa current. (A) Pale blue shaded area shows the conditional OD histogramfor NaV1.5 WT channel opening in the late phase (after 50 ms depolariza-tion), which follows a multiexponential decay. The gray solid line is the ODfit for the WT channel opening in the early phase (reproduced from SI Ap-pendix, Fig. S4E). The red solid line is a scaled OD fit for the IFM/IQMinactivation-deficient mutant opening (B). The blue solid line is a fit for thelate-phase conditional OD by a weighted sum of the early phase and IFM/IQM OD distributions. (B) OD histogram (pale red shaded area) and fit (solidred line) of the IFM/IQM mutant opening. (C–F) Format as in A but forNaV1.5 IQ/AA or S[1904]L mutants at both low and high ambient CaM levels.Gray solid lines are OD fits for the respective mutant channel openings in theearly phase (reproduced from SI Appendix, Figs. S4F and S5E). In all cases, theconditional OD distributions for late channel openings are multiexponential,with the slow component matching the OD distribution of inactivation-deficient (IFM/IQM) channels. (G) Summary of multifaceted modulation ofNaV1.5 by CaM and potential molecular conformational changes. With CaMbound, the carboxy-terminus (CT) interacts with the III-IV linker and preventspremature translocation for the “IFM” inactivation particle to its receptorsite near the S6 domain. Following voltage depolarization and channelopening, the III-IV linker may release from the CT and thereby triggerchannel inactivation. Devoid of apoCaM, allosteric changes may cause the III-IV linker to be released from the CT. This change could result in prematuretranslocation of the “IFM” motif to the S6 receptor site, manifesting asenhanced closed-state inactivation. Alternatively, with a low likelihood, the“IFM” motif may altogether fail to reach its receptor site, resulting in per-sistent channel openings.



Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2





data acquisition interface (InstruTech) driven by custom MATLAB software(MathWorks). Currents were filtered at 5 kHz for single-channel or 2 kHz formultichannel with a four-pole Bessel filter and sampled at 200 kHz. Thesubtraction of capacitive transients were performed using an automatedalgorithm which fit the capacitive transient using convex optimization withL1 regularization. Discrete channel openings were considered as sparseoutliers from the overall multiexponential capacitive transient. We appliedthe following constrained minimization:

mine, s

(12

Imeasured − De − s 2

2 + λ

s 1)s. t. e ≥ 0, s ≤ 0, and s(t<t0) = 0.

Here, Imeasured is the raw input that includes the capacitive transient (Icap)and channel openings, D is a library of exponential functions with amplitude1 and varying time constants, e is the amplitude coefficients associated witheach exponential component, s is a vector representing an estimate ofnegative outliers, that is, channel openings that “corrupt” the desired leakcurrent, and λ is a penalty term that ranged between 0.15 and 0.25. Theactual current trace was obtained by subtracting the multiexponential fitfrom the raw data. For a majority of stochastic records, this leak subtractionalgorithm sufficed. In rare cases, the capacitive transients were manually fit.Unitary current values were estimated using amplitude histograms. Thenumber of channels were estimated by stacking, binomial analysis, andmean-variance analysis. For binomial analysis, we followed an optimizationprocedure, whereby we assumed the number of channels (N∈N, 1 ≤ n ≤ 15)and obtained an estimated PO waveform for each N value. With this esti-mate, the likelihood of observing N openings at each time point is specifiedby the binomial distribution. This theoretical value is compared with anempirical distribution computed from idealized current traces. The numberof channels was estimated as the N value that yielded a minimum leastsquares error between the empirical distribution and binomial estimate andconfirmed using mean-variance analysis.

For first latency and open-duration calculations, stochastic records wereidealized, and sweeps recorded from identical constructs with number ofchannels n = 1 were pooled. It was assumed that data obtained from theidentical construct followed the same statistical parameters, and thereforecombining the data increased the sample size of single-channel analysis.Ensemble average PO waveforms were computed as the average of 80 to 100stochastic records for each voltage. For all experiments, population data wereestimated from at least three independent transfections. For conditionalopenduration measurements from multichannel records, we considered onlyopenings to the one-channel level and also excluded channel openings whoseinitial time of opening was within 50 ms of voltage depolarization.

For multichannel recordings, the unitary current for each patch was es-timated using an amplitude histogram following leak subtraction. Eachstochastic tracewas subsequently idealized. The ensemble average from 50 to100 stochastic traces was computed for each patch and normalized to thepeak value. The average late current for each patch (Rpersist) was computed asthe average normalized PO following 50 ms of depolarization. For PO esti-mation from multichannel recordings, we used mean-variance analysis. Theunitary current amplitude was specified using an amplitude histogram,leaving one free parameter, N, the number of channels in the patch. Forstatistical analysis of peak PO and Rpersist, we used one-way ANOVA followedby Tukey’s multiple comparisons test. Given that Rpersist values followed alog-normal distribution, statistical comparisons were performed with thelogarithm of Rpersist.

Flow Cytometric FRET Two-Hybrid Assay. Flow cytometric FRET assays wereperformed as described (47). Briefly, HEK293 cells (American Type CultureCollection CRL1573) were cultured in 12-well plates and transfected withpolyethylenimine (PEI) 25 kDa linear polymer (Polysciences #2396602). Foreach experiment, we cotransfected the following: 1 μg Cerulean-taggedCaM, 2 μg Venus-tagged NaV1.5 CT construct, and 0.5 μg T antigen. ThecDNA pairs were mixed together in 100 μl of serum-free Dulbecco’s ModifiedEagle Medium media, and 5 μL PEI was added to each sterile tube. Following15 min of incubation, PEI/cDNA mixtures were added to the 12-well plates,and cells were cultured for 2 d prior to experimentation. Protein synthesisinhibitor cycloheximide (100 μM) was added to cells 2 h prior to experi-mentation to halt synthesis of new fluorophores and allow existing fluoro-phores to fully mature. For the determination of Kd,EFF at low Ca2+ levels,

cells were resuspended in Tyrode’s solution containing 0 mM Ca2+ (138 mMNaCl, 4 mM KCl, 1 mM MgCl2, 10 mM Hepes [pH 7.4 using NaOH], 0.2 mMNaHPO4, 1 mM EGTA, and 5 mM D-glucose). For the determination of Kd,EFF

at high Ca2+ levels, cells were resuspended in Tyrode’s solution containing10 mM Ca2+ (138 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM Hepes [pH 7.4using NaOH], 0.2 mM NaHPO4 5 mM D-glucose, and 10 mM CaCl2) and 4 μMionomycin (Sigma-Aldrich) was added 10 min prior to experimentation.

For FRET measurements, we utilized an LSR II (BD Biosciences) flowcytometer equipped with 405, 488, and 633 nm lasers for excitation and 18different emission channels as previously described (47). Forward and sidescatter signals were detected and used to gate for single and healthy cells.For determining FRET efficiency, we measured three distinct fluorescencesignals: 1) Cerulean emission through direct excitation measured throughthe BV421 channel (excitation, 405 nm; emission, 450/50), 2) Venus emissionfrom direct excitation measured via the fluorescein isothiocyanate channel(excitation, 405 nm; dichroic, 505 LP; emission, 525/50), and 3) Venus emis-sion because of FRET via the BV510 channel (excitation, 405 nm; dichroic, 505LP; emission, 525/50). These raw fluorescence measurements are used to obtainVendirect (Venus emission because of direct excitation), Cerdirect (Ceruleanemission because of direct excitation), and FRET efficiency measurements. Flowcytometric signals were collected at a medium flow rate (2,000 to 8,000 events/sec). Fluorescence data were exported as Flow Cytometry Standard 3.0 files forfurther processing and analysis using custom MATLAB functions (MathWorks).Analysis was performed as described (47). Briefly, we calculated the FRET ef-ficiency ED = VenFRET/(VenFRET + fA/fD · Cerdirect). Subsequently, the total con-centration of donors and acceptors were calculated as ND = Cerdirect/(1 − ED)and NA = Vendirect/(gA/gD · fA/fD). The instrument-specific parameters fA/fD andgA/gD are determined on the day of the experiments using a series ofCerulean–Venus dimers with known FRET efficiency. To construct FRET two-hybrid binding curves, we imposed a 1:1 binding isotherm as in previousstudies (46). Specifically, we iteratively fit ED = Emax × [A]free/([A]free + Kd,eff)using the least squares method in which Afree is computed as NA – (ND – Dfree).The free concentration of the donor is computed as the following:

Dfree = 0.5 · (ND–Kd,EFF–NA) + 0.5 ·(ND–Kd,EFF–NA)2 + 4ND ·Kd,EFF

√.

For each FRET pairs, Kd,EFF and ED,max and 95% CIs were obtained by an it-erative constrained least squares fit. For all experiments, we show all indi-vidual cells obtained from at least two independent transfections.

Computational Ventricular Action Potential Modeling. Computational modelingwas performed with the ToR-ORd model simulated using MATLAB (56). Weadjusted the model parameters to reflect the kinetic profile of the NaV1.5 POwaveform from our experiments both in the presence and absence of CaM, andthe amplitude of the persistent current and PO/peak values were constrained tomatch the empirical relationship obtained in Fig. 5K. Specifically, we adjusted

τm as follows: τm = 1=(4.228125eV+11.6434.77 + 5.345e−V+77.42

5.95 ). Furthermore, the peak Na

current conductance was adjusted as follow: GNa = 11.7802 · (0.3 + 0.7*fb|CaM),while the late Na current conductance was modified as GNa,L =0.014 · (36− 35*fb|CaM) · (0.3 + 0.7*fb|CaM). For simulations accounting for the

effect of FGF12/13, the peak Na current conductance was adjusted as above.However, the late Na current conductance was modified as GNa,L =0.014 · (0.3+ 0.7*fb|CaM) to maintain the overall ratio of peak to late Na

conductance. Action potentials were simulated for 100 beats at variousheart rates, and only the final two beats were considered for the mea-surement of APD.

Data Availability.All study data are included in the article and/or SI Appendix.

ACKNOWLEDGMENTS. We thank Dr. Richard Aldrich, Dr. L. Mario Amzel,Dr. Sandra Gabelli, Dr. Henry Colecraft, Dr. Steven O. Marx, Dr. Filip vanPetegem, and Dr. Ivy Dick for valuable insight and feedback on this work.This study is supported by funding from National Heart, Lung, and BloodInstitute and National Institute of Neurological Disorders and Stroke.Research supported in this publication was performed in the ColumbiaCenter for Translational Immunology flow cytometry core, supported in partby the Office of the Director, NIH, under award S10RR027050. The content issolely the responsibility of the authors and does not necessarily representthe official views of the NIH.

1. B. Hille, Ionic Channels of Excitable Membranes (Sinauer Associates, Sunderland, MA,

1984), pp. 226–248.2. W. A. Catterall, M. J. Lenaeus, T. M. Gamal El-Din, Structure and pharmacology of voltage-

gated sodium and calcium channels. Annu. Rev. Pharmacol. Toxicol. 60, 133–154 (2020).

3. T. Zimmer, R. Surber, SCN5A channelopathies–An update on mutations and mecha-

nisms. Prog. Biophys. Mol. Biol. 98, 120–136 (2008).4. H. Abriel, J. S. Rougier, J. Jalife, Ion channel macromolecular complexes in car-

diomyocytes: Roles in sudden cardiac death. Circ. Res. 116, 1971–1988 (2015).



PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2



5. E. Wan et al., Aberrant sodium influx causes cardiomyopathy and atrial fibrillation inmice. J. Clin. Invest. 126, 112–122 (2016).

6. C. A. Remme, Cardiac sodium channelopathy associated with SCN5A mutations:Electrophysiological, molecular and genetic aspects. J. Physiol. 591, 4099–4116 (2013).

7. A. A. M. Wilde, A. S. Amin, Clinical spectrum of SCN5A mutations: Long QT syndrome,Brugada syndrome, and cardiomyopathy. JACC Clin. Electrophysiol. 4, 569–579 (2018).

8. P. B. Bennett, K. Yazawa, N. Makita, A. L. George Jr, Molecular mechanism for aninherited cardiac arrhythmia. Nature 376, 683–685 (1995).

9. J. R. Bankston et al., A novel LQT-3 mutation disrupts an inactivation gate complex withdistinct rate-dependent phenotypic consequences. Channels (Austin) 1, 273–280 (2007).

10. C. Antzelevitch et al., Brugada syndrome: A decade of progress. Circ. Res. 91,1114–1118 (2002).

11. C. Antzelevitch, The Brugada syndrome: Ionic basis and arrhythmia mechanisms.J. Cardiovasc. Electrophysiol. 12, 268–272 (2001).

12. R. S. Kass, Sodium channel inactivation in heart: A novel role of the carboxy-terminaldomain. J. Cardiovasc. Electrophysiol. 17, S21–S25 (2006).

13. J. D. Kapplinger et al., An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetictesting. Heart Rhythm 7, 33–46 (2010).

14. F. Van Petegem, P. A. Lobo, C. A. Ahern, Seeing the forest through the trees: Towardsa unified view on physiological calcium regulation of voltage-gated sodium channels.Biophys. J. 103, 2243–2251 (2012).

15. G. S. Pitt, S. Y. Lee, Current view on regulation of voltage-gated sodium channels bycalcium and auxiliary proteins. Protein Sci. 25, 1573–1584 (2016).

16. S. B. Gabelli, J. B. Yoder, G. F. Tomaselli, L. M. Amzel, Calmodulin and Ca(2+) controlof voltage gated Na(+) channels. Channels (Austin) 10, 45–54 (2016).

17. J. C. Mulley et al., SCN1A mutations and epilepsy. Hum. Mutat. 25, 535–542 (2005).18. F. K. Satterstrom et al.; Autism Sequencing Consortium; iPSYCH-Broad Consortium,

Large-scale exome sequencing study implicates both developmental and functionalchanges in the neurobiology of autism. Cell 180, 568–584.e23 (2020).

19. S. J. Sanders et al., Progress in understanding and treating SCN2A-mediated disorders.Trends Neurosci. 41, 442–456 (2018).

20. U. B. S. Hedrich, S. Lauxmann, H. Lerche, SCN2A channelopathies: Mechanisms andmodels. Epilepsia 60, S68–S76 (2019).

21. M. H. Meisler et al., SCN8A encephalopathy: Research progress and prospects. Epi-lepsia 57, 1027–1035 (2016).

22. R. Horie et al., EF hand-like motif mutations of Nav1.4 C-terminus cause myotonicsyndrome by impairing fast inactivation. Muscle Nerve 61, 808–814 (2020).

23. S. C. Cannon, Sodium channelopathies of skeletal muscle. Handb. Exp. Pharmacol.246, 309–330 (2018).

24. M. D. Feldkamp, L. Yu, M. A. Shea, Structural and energetic determinants of apocalmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodiumchannel. Structure 19, 733–747 (2011).

25. B. Chagot, W. J. Chazin, Solution NMR structure of Apo-calmodulin in complex withthe IQ motif of human cardiac sodium channel NaV1.5. J. Mol. Biol. 406, 106–119(2011).

26. M. Mori, T. Konno, T. Morii, K. Nagayama, K. Imoto, Regulatory interaction of sodiumchannel IQ-motif with calmodulin C-terminal lobe. Biochem. Biophys. Res. Commun.307, 290–296 (2003).

27. B. R. Gardill, R. E. Rivera-Acevedo, C. C. Tung, F. Van Petegem, Crystal structures ofCa2+-calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain inbinding and inactivation. Proc. Natl. Acad. Sci. U.S.A. 116, 10763–10772 (2019).

28. S. B. Gabelli et al., Regulation of the NaV1.5 cytoplasmic domain by calmodulin. Nat.Commun. 5, 5126 (2014).

29. J. B. Yoder et al., Ca2+-dependent regulation of sodium channels NaV1.4 and NaV1.5 iscontrolled by the post-IQ motif. Nat. Commun. 10, 1514 (2019).

30. C. Wang, B. C. Chung, H. Yan, S. Y. Lee, G. S. Pitt, Crystal structure of the ternarycomplex of a NaV C-terminal domain, a fibroblast growth factor homologous factor,and calmodulin. Structure 20, 1167–1176 (2012).

31. C. Wang et al., Structural analyses of Ca2+/CaM interaction with NaV channelC-termini reveal mechanisms of calcium-dependent regulation. Nat. Commun. 5, 4896(2014).

32. M. Ben-Johny et al., Conservation of Ca2+/calmodulin regulation across Na and Ca2+channels. Cell 157, 1657–1670 (2014).

33. I. Deschênes et al., Isoform-specific modulation of voltage-gated Na(+) channels bycalmodulin. Circ. Res. 90, E49–E57 (2002).

34. J. Kim et al., Calmodulin mediates Ca2+ sensitivity of sodium channels. J. Biol. Chem.279, 45004–45012 (2004).

35. M. F. Sarhan, C. C. Tung, F. Van Petegem, C. A. Ahern, Crystallographic basis forcalcium regulation of sodium channels. Proc. Natl. Acad. Sci. U.S.A. 109, 3558–3563(2012).

36. Z. Wang et al., Calmodulin binds to the N-terminal domain of the cardiac sodiumchannel Nav1.5. Channels (Austin) 14, 268–286 (2020).

37. C. N. Johnson et al., A mechanism of calmodulin modulation of the human cardiacsodium channel. Structure 26, 683–694.e3 (2018).

38. B. R. Gardill et al., The voltage-gated sodium channel EF-hands form an interactionwith the III-IV linker that is disturbed by disease-causing mutations. Sci. Rep. 8, 4483(2018).

39. A. R. Gade, S. O. Marx, G. S. Pitt, An interaction between the III-IV linker and CTD inNaV1.5 confers regulation of inactivation by CaM and FHF. J. Gen. Physiol. 152,e201912434 (2020).

40. C. H. Peters, A. R. Watkins, O. L. Poirier, P. C. Ruben, E1784K, the most commonBrugada syndrome and long-QT syndrome type 3 mutant, disrupts sodium channelinactivation through two separate mechanisms. J. Gen. Physiol. 152, e202012595(2020).

41. H. Shen et al., Structure of a eukaryotic voltage-gated sodium channel at near-atomicresolution. Science 355, eaal4326 (2017).

42. T. Clairfeuille et al., Structural basis of α-scorpion toxin action on Nav channels. Sci-ence 363, eaav8573 (2019).

43. I. W. Glaaser et al., Perturbation of sodium channel structure by an inherited long QTsyndrome mutation. Nat. Commun. 3, 706 (2012).

44. H. Yan, C. Wang, S. O. Marx, G. S. Pitt, Calmodulin limits pathogenic Na+ channelpersistent current. J. Gen. Physiol. 149, 277–293 (2017).

45. A. O. Verkerk, A. S. Amin, C. A. Remme, Disease modifiers of inherited SCN5Achannelopathy. Front. Cardiovasc. Med. 5, 137 (2018).

46. S. R. Lee, L. Sang, D. T. Yue, Uncovering aberrant mutant PKA function with flowcytometric FRET. Cell Rep. 14, 3019–3029 (2016).

47. S. Rivas, K. Hanif, N. Chakouri, M. Ben-Johny, “Probing ion channel macromolecularinteractions using fluorescence resonance energy transfer” in Methods in Enzymol-ogy, D. L. Minor Jr., H. M. Colecraft, Eds. (Academic Press, 2021).

48. S. Biswas, D. DiSilvestre, Y. Tian, V. L. Halperin, G. F. Tomaselli, Calcium-mediateddual-mode regulation of cardiac sodium channel gating. Circ. Res. 104, 870–878(2009).

49. C. G. Vanoye, C. Lossin, T. H. Rhodes, A. L. George Jr, Single-channel properties ofhuman NaV1.1 and mechanism of channel dysfunction in SCN1A-associated epilepsy.J. Gen. Physiol. 127, 1–14 (2006).

50. R. Albitz, G. Droogmans, B. Nilius, The conductance of single cardiac sodium channelsfrom guinea pig depends on the intracellular sodium concentration. Biochim. Bio-phys. Acta 1068, 254–256 (1991).

51. M. Ben Johny, P. S. Yang, H. Bazzazi, D. T. Yue, Dynamic switching of calmodulininteractions underlies Ca2+ regulation of CaV1.3 channels. Nat. Commun. 4, 1717(2013).

52. R. Banerjee et al., Bilobal architecture is a requirement for calmodulin signaling toCaV1.3 channels. Proc. Natl. Acad. Sci. U.S.A. 115, E3026–E3035 (2018).

53. J. Abrams et al., Fibroblast growth factor homologous factors tune arrhythmogeniclate NaV1.5 current in calmodulin binding-deficient channels. JCI Insight 5, 141736(2020).

54. S. Kellenberger, J. W. West, T. Scheuer, W. A. Catterall, Molecular analysis of theputative inactivation particle in the inactivation gate of brain type IIA Na+ channels.J. Gen. Physiol. 109, 589–605 (1997).

55. H. A. Hartmann, A. A. Tiedeman, S. F. Chen, A. M. Brown, G. E. Kirsch, Effects of III-IVlinker mutations on human heart Na+ channel inactivation gating. Circ. Res. 75,114–122 (1994).

56. J. Tomek et al., Development, calibration, and validation of a novel human ventric-ular myocyte model in health, disease, and drug block. eLife 8, e48890 (2019).

57. J. A. Hennessey et al., FGF12 is a candidate Brugada syndrome locus. Heart Rhythm 10,1886–1894 (2013).

58. M. Ben-Johny et al., Towards a unified theory of calmodulin regulation (calm-odulation) of voltage-gated calcium and sodium channels. Curr. Mol. Pharmacol. 8,188–205 (2015).

59. T. L. Wingo et al., An EF-hand in the sodium channel couples intracellular calcium tocardiac excitability. Nat. Struct. Mol. Biol. 11, 219–225 (2004).

60. B. Chagot, F. Potet, J. R. Balser, W. J. Chazin, Solution NMR structure of the C-terminalEF-hand domain of human cardiac sodium channel NaV1.5. J. Biol. Chem. 284,6436–6445 (2009).

61. V. N. Shah et al., Calcium-dependent regulation of the voltage-gated sodium channelhH1: Intrinsic and extrinsic sensors use a common molecular switch. Proc. Natl. Acad.Sci. U.S.A. 103, 3592–3597 (2006).

62. N. M. Ashpole et al., Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulatescardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol.Chem. 287, 19856–19869 (2012).

63. D. L. Minor Jr, F. Findeisen, Progress in the structural understanding of voltage-gatedcalcium channel (CaV) function and modulation. Channels (Austin) 4, 459–474 (2010).

64. J. Sun, R. MacKinnon, Cryo-EM structure of a KCNQ1/CaM complex reveals insightsinto congenital long QT syndrome. Cell 169, 1042–1050.e9 (2017).

65. P. W. Kang et al., Calmodulin acts as a state-dependent switch to control a cardiacpotassium channel opening. Sci. Adv. 6, eabd6798 (2020).

66. D. Gong et al., Modulation of cardiac ryanodine receptor 2 by calmodulin. Nature572, 347–351 (2019).

67. D. M. Bers, Macromolecular complexes regulating cardiac ryanodine receptor func-tion. J. Mol. Cell. Cardiol. 37, 417–429 (2004).

68. M. Abdelsayed et al., Differential calcium sensitivity in NaV 1.5 mixed syndromemutants. J. Physiol. 595, 6165–6186 (2017).

69. F. Potet, T. M. Beckermann, J. D. Kunic, A. L. George Jr, Intracellular calcium atten-uates late current conducted by mutant human cardiac sodium channels. Circ. Ar-rhythm. Electrophysiol. 8, 933–941 (2015).

70. D. Jiang et al., Structure of the cardiac sodium channel. Cell 180, 122–134.e10 (2020).71. F. Potet et al., Functional interactions between distinct sodium channel cytoplasmic

domains through the action of calmodulin. J. Biol. Chem. 284, 8846–8854 (2009).



Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

2


Elementary mechanisms of calmodulin regulation of NaV1.5 ...

Documents

Transcript of Elementary mechanisms of calmodulin regulation of NaV1.5 ...