ION CHANNELS, RECEPTORS AND TRANSPORTERS

The bundle crossing region is responsible for the inwardlyrectifying internal spermine block of the Kir2.1 channel

Chiung-Wei Huang & Chung-Chin Kuo

Received: 29 January 2013 /Revised: 3 May 2013 /Accepted: 24 June 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Inward rectifier potassium channels conduct K+

across the cell membrane more efficiently in the inward thanoutward direction in physiological conditions. Voltage-dependent and flow-dependent blocks of outward K+ cur-rents by intracellular polyamines (e.g., spermine (SPM))have been proposed as the major mechanisms underlyinginward rectification. In this study, we show that the SPMblocking affinity curve is shifted according to the shift in K+

reversal potential. Moreover, the kinetics of SPM entry toand exit from the binding site are correlatively slowed byspecific E224 and E299 mutations, which always also dis-rupt the flux coupling feature of SPM block. The entry ratescarry little voltage dependence, whereas the exit rates are e-fold decelerated per ∼15 mV depolarization. Interestingly,the voltage dependence remains rather constant among WTand quite a few different mutant channels. This voltagedependence offers an unprecedented chance of mapping thelocation (electrical distance) of the SPM site in the porebecause these kinetic data were obtained along the prepon-derant direction of K+ current flow (outward currents for theentry rate and inward currents for the exit rate) and thuscontamination from flow dependence should be negligible.Moreover, double mutations involving E224 and A178 orM183 seem to alter the height of the same asymmetrical

barrier between the SPM binding site and the intracellularmilieu. We conclude that the SPM site responsible for theinward rectifying block is located at an electrical distanceof ∼0.5 from the inside and is involved in a flux couplingsegment in the bundle crossing region of the pore. Withpreponderant outward K+ flow, SPM is “pushed” to theoutmost site of this segment (∼D172). On the other hand,the blocking SPM would be pushed to the inner end of thissegment (∼M183–A184) with preponderant inward K+ flow.Moreover, E224 and E299 very likely electrostatically inter-act with the other residues (e.g., R228, R260) in the cyto-plasmic domain and then allosterically keep the bundlecrossing region in an open conformation appropriate for theflux coupling block of SPM.

Keywords Inwardly rectifier K+ channel . Flow-dependentblocking . Bundle crossing region . Gating . Permeation

Introduction

Inward rectifier K+ channel (Kir channels) constitute a familyof K+ channels composed of two transmembrane segments(2TM) channels [21, 30, 32]. Inward rectifier potassium chan-nels conduct K+ across the cell membrane much faster in-wardly than outwardly [14–16, 22]. This intriguing conduc-tion property is essential for the biological function of thesechannels, including maintenance of the resting membranepotential close to the K+ equilibrium potential without losingtoo much intracellular K+ to the extracellular compartmentduring membrane depolarization [10, 14, 27, 54]. Abnormalactivities of Kir channels have been linked to a variety ofcardiac, endocrine, and neurological diseases [58, 66],including abnormal electrolyte processing by the kidney,cardiac arrhythmia, seizures, Andersen syndrome, and Batter

Electronic supplementary material The online version of this article(doi:10.1007/s00424-013-1322-0) contains supplementary material,which is available to authorized users.

C.<W. Huang :C.<C. Kuo (*)Department of Physiology, National Taiwan University College ofMedicine, No. 1, Jen-Ai Road, 1st Section, Taipei 100, Taiwane-mail: chungchinkuo@ntu.edu.tw

C.<C. KuoDepartment of Neurology, National Taiwan University Hospital,Taipei, Taiwan

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syndrome [19, 58–60, 66]. Elucidation of the molecular oper-ation of Kir channels thus may be of both physiological andpathophysiological significance.

The molecular mechanisms underlying internal spermine(SPM)-related rectification in Kir channels have been exten-sively investigated, most focusing on a pore-blocking model[12, 13, 24, 25, 35, 42, 43, 49, 50, 57]. However, datacollected from studies for the past decades indicate that theWoodhull model [67] is deficient in the description of SPMblock in this channel. For instance, the electrical distance (δ)of SPM binding site has been reported from 0.3 to 1.6 [12, 24,25, 41, 49]. In addition, the Woodhull model predicts a linearrelationship between the semilogarithm of dissociation con-stant and membrane voltage and also K+ or SPMconcentration-independent estimates of the SPM binding af-finity or the electrical distance of the SPM binding site.However, previous findings contradictory to the predictionshave been reported [12, 13, 44, 56, 57, 62]. Because theelectrical distance defines the location of an ionic site in thepore in terms of the percentage of total voltage drop across thecell membrane, the summation of the electrical distance frominside or outside should not exceed 1 for any ionic block. Theestimated electrical distance of ≥1, along with the nonlinearrelationship between the semilogarithm of SPMKd and mem-brane voltage [12, 24, 25, 49], and the K+ or SPM concentra-tion dependence of SPM Kd [44, 61, 62], would altogethersuggest a more complex situation such as strong flux couplingeffect rather than a simple voltage-dependent pore-blockingphenomenon. The “pure” voltage dependence or electricaldistance without contamination from the flux coupling effect,however, has remained uncharacterized.

In the family of the Kir channels, the Kir2.1 channelshows the strongest rectifying feature in the current–volt-age (i–V) relation [1, 45, 47–51]. The single channel con-ductance of Kir2.1 inward currents is ∼40 pS (slope con-ductance measured at Vm=−140 to −20 mV) in bothXenopus oocytes [5, 12, 41] and mammalian cell lines[49, 50, 53]. Also, the single channel conductance of theinwardly rectifying K+ currents is ∼40 pS in excitable cellssuch as cardiomyocytes [47, 48]. E224 and E299, twoacidic residues located in the cytoplasmic domain internalto the M2 segment in the inner vestibule of Kir2.1 channelpore, may be involved in the control of ion conductancebecause of electrostatic and surface charge effects [6, 12,68, 69, 73]. Mutations involving E224 and E299 signifi-cantly reduced the susceptibility of Kir2.1 channels tointernal SPM [9, 12, 28, 29, 53, 63, 68, 71] and alsomarkedly diminished the flow-dependent features of SPMblock [12, 29, 36, 53, 56, 57]. However, E224 is located inthe cytoplasmic domain according to the result of the X-raycrystallography [18, 55, 64]. This is a very wide part of thepore (the radius is ∼15 Ǻ), presumably too wide to serve asingle-file pore region. The residues directly constituting

the flux coupling segment and the key biophysical or func-tional attributes of the SPM blocking site are, therefore, notfully clear.

According to the crystallographic data of the Kir2.2channel, residues I176 to K185 constitute the bundle cross-ing region [18, 64], which could also be involved in theblocking effect of SPM on the Kir2.1 channel [4, 37–40, 52,56, 70]. Moreover, D172 has been proposed to constitutethe SPM blocking site [13, 25, 35, 38–40, 50, 57]. If theleading end of SPM lies near D172, then its trailing endmay lie in or near the bundle crossing region in the trans-membrane domain [7]. However, the molecular interactionsbetween SPM and its blocking site or the flux couplingsegment, and the functional role of the bundle crossingregion in ion permeation of the Kir2.1 channel, haveremained largely unexplored. We therefore endeavored toinvestigate how the bundle crossing region contributes tothe inward rectifying SPM block and the other molecularbehavior of the Kir2.1 channel and found that residuesA178 and M183 very likely directly constitute the fluxcoupling pore segment containing the SPM blocking sitelocated at electrical distance of ∼0.5 from inside. Moreover,E224 and E299 probably electrostatically interact withR228 and R260 to play a pivotal role in the “open-closing”or gating-like conformational changes in the bundle cross-ing region of the pore.

�Fig. 1 Inhibition of WT Kir2.1 currents by different concentrations ofinternal SPM in 100 or 20 mM external K+. a Sample sweeps demonstrat-ing inhibition ofWTKir2.11 currents in the same patch by internal SPM insymmetrical 100 mM K+. b The relative currents are defined by the ratiobetween the steady-state currents either in SPM or in control at eachdesignated potential and are plotted against SPM concentration (n=5–8).The data obtained at different voltages (e.g., –30, –10, +10, +30, and+50 mV) are fitted by Eq. 1 (see “Materials and methods”). c The Kd frompart b (for voltages between −30 and +50mV) is plotted against voltage ina semilogarithmic scale. The Kd−voltage relationship is clearly nonlinear,and there is a dramatic decrease in the Kd value from −30 to +30 mV(i.e., ±30 mV from the 0 mV, K+ reversal potential). At voltage morepositive than +20 mV, Kd reaches ∼10−4 μM and becomes essentiallyvoltage independent, probably signaling outward exit of the blocking SPMat these positive potentials. The IR index (the ratio between Kd valuesat −30 and +30 mV) is ∼102,000 in this case. The blocking effect of theinternal SPM thus is closely correlated with the direction of preponderantcurrent flow or is strongly flow dependent. d The same experiments anddata analyses as that in parts a to c were repeated in 20 mM external K+

(the 100-mM internal K+ remains unchanged) (n=5–10). The dataobtained at different voltages (e.g., −100, −60, −20, +20, and +50 mV)are fitted by Eq. 1 (see “Materials and methods”). e The Kd–voltagerelationship in external 20/internal 100 mM K+ remains similar to that insymmetrical 100 mM K+, but is shifted by ∼−40 mV in the voltage axis.The dramatic decrease of Kd in this case happens roughly between −70and −10 mV (±30 from −40 mV, the K+ reversal potential). The solid linedenotes the data in symmetrical 100 mMK+ channel (taken from part c forcomparison). Also note that the Kd at +30∼+50 mV is higher in 20/100 mM K+ than that in symmetrical 100 mM K+

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Materials and methods

Molecular biology and preparation of Xenopus oocytes

Mouse macrophage Kir2.1 DNA subcloned into BluescriptII SK1 was a kind gift from Dr. Ru-Chi Shieh (AcademicSinica, Taiwan). Mutant channel cDNAs were prepared

using the QuickChange site-directed mutagenesis kit(Stratagene, La Jolla, CA, USA). Each mutant DNA ofKir2.1 channels was confirmed by an automatic DNA se-quencer. cRNAwas made from purified NotI-linearizd cDNAwith in vitro T7 transcription reactions (mMESSAGEmMACHINE, Ambion, Austin, TX, USA) from NotI-linearized cDNA of Kir2.1 channels. This study was carried

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out in strict accordance with the recommendations in theGuide for the Care and Use of Laboratory Animals of theNational Institutes of Health. All animal experiments wereperformed in accordance with animal welfare guidelines andwere approved by the Institutional Animal Care and UseCommittee of National Taiwan University College of Medi-cine. The experiments in this study were conducted in accor-dance with policies and regulations given by Drummond [8].Xenopus oocytes were obtained by partial ovariectomy fromfrogs anesthetized with 0.1 % tricaine, defolliculated using2 % collagenase and then maintained at 18 °C in ND96solution which contains (in millimolar) 96 NaCl, 2 KCl, 1.8MgCl2, 1.0 CaCl2, 5 HEPES, as well as 20 μg gentamicin/ml(pH 7.6). Oocytes were pressure injected with Kir2.1 cRNA24 h after defolliculation and were used for electrophysiolog-ical recordings within 1–7 days.

Electrophysiological recordings

Macroscopic currents were recorded from inside-out patchesfrom Xenopus oocytes expressing wild type or mutant Kir2.1channels with an Axopatch 200A amplifier (Axon Instru-ments, Inc.). The pipettes were pulled from borosilicatemicropipettes and fire polished. The pipette resistance was100∼500 kΩ when filled with extracellular recording solu-tions. Unless otherwise specified, the extracellular recordingand intracellular (bath) solutions both contained (in millimo-lar): 67.3 KCl, 5 K2EDTA, 8 K2HPO4, 2 KH2PO4, and 4.7KOH (the “100 mM K+ solution”), pH 7.4. The bath solu-tions also contained 25 μML-α-phosphatidylinositol-4,5-bisphosphate (PIP2) and 0∼10 mM SPM (both from SigmaChemical Co., St. Louis, MO). For the experiments in 20 or4 mM external K+, the intracellular solution was notchanged, but the extracellular solution contained 20 and4 mM, respectively. For the experiments in different inter-nal pH, the intracellular was titrated to pH 6.4 and 8.4,respectively, whereas the extracellular solution remainedunchanged (pH 7.4). The inside-out patch was moved infront of an array of square glass emitting the 100 mM K+

intracellular solution and thus subject to continuous perfu-sion of the intracellular solution for at least 5 min to washout potential intracellular endogenous blockers before ac-tual experiments were carried out. Data were filtered at1∼2 kHz and digitized at 1∼10 kHz (Digidata 1200A;Axon Instruments, Inc.). pClamp 6.0 software was used foramplifier control and data acquisition. Unless otherwise speci-fied, the voltage across the patch membrane was firsthyperpolarized from the 0-mV holding potential to −100 mVfor 10 ms and then stepped to different test voltages be-tween −150 and 100 mV for ∼100 ms in 10 mV increment.The steady-state current amplitude was given by the averagecurrent at the last 5 ms in the test pulse, which had alwaysreached an apparent plateau.

A three-barrel square glass tube, enabling quick changebetween two different solutions each time, was used tostudy the kinetics of methanethiosulfonate (MTS) modifi-cation in Fig. 4c. An inside-out patch was established using100 mM K+ intracellular solutions. The patch was thenmoved in front of the three-barrel square glass tube (internaldiameter ∼0.6 mm) emitting different intracellular record-ing solutions. The glass tube holder was connected to astepper control (SF-77B perfusion system, Warner Instru-ments) which enabled rapid switch between different glasstubes and thus rapid solution change (50 % solution exchangetime ∼40 ms [2, 3, 34, 72]).

MTSEA modification

The reagent methanethiosulfonate ethylammonium (MTSEA)(Toronto Research Chemical, Inc., Ontario, Canada) was pre-pared daily, stored in ice-cold conditions, and then mixed intothe solutions to reach a final concentration of 100 μM imme-diately prior to use. Modification rates were determined by the

�Fig. 2 The emergence of a slow component of internal SPM unblock inE224G mutant channels. a The Kd of E224G and E224Q mutant chan-nels are plotted against voltage (the same approach as that in Fig. 1c,n=5–8). The change in Kd around 0 mV (the reversal potential of K+) ismuch less dramatic in the pressure of E224G and especially E224Qmutations. The WT channel data (solid line) are taken from Fig. 1c forcomparison. b Comparison of the IR indices of internal SPM blockamong the WT, E22G, and E224Q mutant channels (which are∼102,000, ∼22.3, and ∼2.6, respectively). c The patch which expressedE224G mutant channel was held at 0 mVand stepped twice to −100 mV.The intervening gap between the two pulses was set at +100 mV andlengthened by 10 ms between each sweep. The sample sweeps show aslow tail only when the patch was repolarized from a positive (e.g.,+100 mV) to a negative potential (e.g., –100 mV) in the presence ofinternal SPM, but not in control. Interestingly, the slow tail gets largerwith the lengthening of the +100-mV pulse (until a plateau is reached),but the decay kinetics remains rather constant. The development of theslow tail in the second −100 mV pulse is used as a measure of SPMbinding to this slow unbinding site at +100 mVand is indeed faster in 100than in 3 μM (the dotted lines are the monoexponential fit to the growingamplitude of the slow tail and have time constants of∼180 and∼20ms for3 and 100 μM SPM, respectively). Note that the course of slow tailgrowing is well correlated with the decay of the outward currents (seealso part d) and that the slow tails always reach the same maximalmagnitude plateau in different concentrations of SPM. Moreover, thereis always a fast phase of the inward currents preceding the slow tail uponmembrane hyperpolarization (i.e., when the currents are just turninginward from outward). dCumulative results are obtained from six patcheswith the same protocol describe in part c for the E224G mutant channel.The tauon, 1 and tauon, 2 denote the time constant of the growing course ofthe slow tails (at −100 mV) and the time constant of the decay of themacroscopic outward currents (at +100 mV) in 100 μM SPM in part c,respectively. e Cumulative results are obtained from six patches with thesame protocol described in part c for the E224G mutant channel. Theratios between the amplitude of the fast and slow phases of the inwardcurrents upon stepping to −100 from +100 mV (see also part c) areessentially the same in different SPM concentrations (3, 10, 30, and100 μM)

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change of current amplitude in the presence of MTSEA re-agent for different length of time.

Data analysis

The susceptibility of wild type (WT) and mutant channels tointernal SPM was examined based on apparent Kd values. In

brief, the dose–response relationships of the SPM block ofKir2.1 were fitted using the modified Hill equation [11, 12,24, 26, 41, 71]. We found that the data of steady-state SPMblock could be well fitted with the Hill equation (a one-to-one binding reaction) at membrane potentials more positivethan ∼−30 mV in symmetrical 100 mM K+ or −100 mV in20 mM external/100 mM internal K+ (Eq. 1)

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f ¼ 1

1þ SPM½ �Kd

ð1Þ

In Eq. 1, f is the relative current (the ratio between thesteady-state current amplitude in SPM and that in control),[SPM] is the concentration of SPM, and Kd is the apparentdissociation constant of SPM. Figure 1a shows inhibition ofmacroscopic WT Kir2.1 currents in symmetrical 100 mMK+ by 0.1 and 1.0 μM intracellular SPM. The outwardcurrents are inhibited by SPM with accelerated decay in adose-dependent fashion, whereas the inward currents arenot evidently affected by SPM. Figure 1b plots the relativecurrents versus SPM concentration at different voltages.Figure 1c plots Kd against membrane voltage in a semilog-arithmic scale. To facilitate the comparison of flow depen-dence in different channels, we arbitrarily define the ratiobetween the Kd values at −30 and +30 mV from the reversal

Fig. 3 The binding and unbinding kinetics of internal SPM in E224Gmutant channels. a The reciprocals of the time constants (on rate) for thedevelopment of the slow tail in Fig. 2c are plotted against the SPMconcentration (the depolarization pulses were set between +90 to+120 mV). The dotted lines are linear fits with Y-intercept and slope of6.4 s−1 and 5.0×105 M−1 s−1 (+90 mV); 9.2 s−1 and 5×105 M−1 s−1

(+100 mV); 10.3 s−1 and 5×105 M−1 s−1 (+110 mV); and 11 s−1 and6×105 M−1 s−1 (+120 mV), respectively (n=5–10). b The slopes in part aare plotted against voltage and fitted with a regression line of theform:kon=2.8×10

5 exp (0.15 V/25 mV) M−1 s−1, where V is the mem-brane voltage in millivolt. The regression line indicates an equivalentelectrical distance (δ) of ∼0.04 for the SPM binding site (with thepresumption of four effective charges on SPM). c The inverses of therelaxation time constants of the slow tail currents are plotted against

membrane potential in semilogarithmic scale for the E224G mutantchannels (n=5). The second negative pulse in the same two-pulse proto-col was set to −50∼−100 mV to examine the voltage dependence of SPMunblock from the binding site. The data are fitted with the followingequation off rate(V)=off rate(0) exp(−ZδV/25 mV), where V is the mem-brane potential in millivot, and Z and δ are the charges on the blocker andthe equivalent electric distance of the binding site from inside, respec-tively. The off rate(0) and Zδ are ∼0.1 s−1 and 2.3 for the E224G mutantchannels. d The reciprocals of the time constants of development of slowtail currents (on rate at +100 mV) are plotted against the decay timeconstants of the slow tail (off rate at −100 mV) for each of the E224mutant (E224Q, E224A, E224C, and E224G) channel. The dashed line isa linear regression demonstrating the strong linear correlation between thetwo parameters (n=5–7)

�Fig. 4 Elimination of the extremely slow component of SPM unblock inE224Q mutant channel by concomitant mutations at A178 and M183. aSample sweeps of the extremely slow tail currents in the E224Q mutantchannel in symmetrical 100 mMK+ (see Fig. 2c for the pulse protocol). Theslow tails are much slower than that in the E224Gmutant channel. Also notethat higher concentrations of SPM are needed to observe the slow tails. Theextremely slow tails signaling SPM unbinding in the E224Q single mutantchannel, however, are greatly accelerated or even no longer discernible in theE224Q/A178C, E224Q/A178T, and E224Q/M183W double mutant chan-nels. b Cumulative results are obtained for the E224Q, E224Q/A178C,E224Q/A178T, and E224Q/M183W double mutant channels (each n=5).The tauon, 1 and tauon, 2 denote the time constant of the growing course of theslow tails (at−100mV) and the time constant of the decay of themacroscopicoutward currents (at +100 mV) in 300 μM SPM in part a, respectively (thesame approach as that in Fig. 2d). c Cumulative results are obtained from thetime constants of MTSEA modification in the WT and different mutantchannels at a holding potential of +40 mV. The inverses of the time constantsof MTSEA modification (the modification rates) are 19.7±7.7, 14.6±3.2,15.6±2.9, 0.06±0.01, 1.3±0.3, and 0.84±0.21 s−1 for the WT, A178T,M183W, E224Q, E224Q/A178T, and E224Q/M183W mutant channels,respectively (n=3–9). d The IR index is decreased in the A178C, M183W,M183N, A184Q, and especially in the A184R mutant channels

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potential of K+ (EK+) as “inward rectification (IR) index”.

All data are expressed as mean ± SEM.

Homology modeling of Kir2.1 channel

Homology models of the mouse Kir2.1 channel were builtbased on the coordinates from the X-ray crystal structure of

the mouse Kir2.1 and chicken Kir2.2 channel [18, 55, 64].Specifically, the structure of transmembrane domain wascreated through homology modeling based on the structuretemplate of the chicken Kir2.2 channel (PDB ID, 3SPI)according to the BLAST search results, and then, the struc-ture of cytoplasmic domain was also created with a templatefrom the mouse Kir2.1 channel (PDB ID, 1U4F). The

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aligned sequences were presented to the Discovery Studiov2.5 program (DS v2.5) (Accelrys Inc., San Diego, CA) togenerate the secondary structure and relative positions of thechosen residues of the Kir2.1 model. We first chose thestructural models with the lowest free energy potentials.Upon completion of the initial operations, overall energyminimization was performed again to obtain the final modelof the Kir2.1 channel.

Results

Effects of reversal potential shift and K+ ions on internalSPM block in WT Kir2.1 channels

We investigated the flow dependence of SPM block. Inview of the possibility of interaction between SPM andpermeating K+, we examined the effects of the internalSPM in symmetrical 100 mM K+, in higher but still sym-metrical (300 mM) K+, and in asymmetrical K+ (20/100 or20 mM external and 100 mM internal K+, reversalpotential=−40 mV) (Figs. 1d, e and S1). Similar to the casein symmetrical 100 mMK+, the Kd in symmetrical 300 mMK+ changes sharply around 0 mV (i.e., between −30 and +30 mV) with an IR index of ∼31,000 (Fig. S1). The absolutevalues of Kd, interestingly, are always ∼100-fold largerthan those in symmetrical 100 mM K+ at every voltage.On the other hand, the Kd in asymmetric (20/100 mM) K+

changes sharply around −40 mV. We also carried out theexperiments in 4 mM extracellular K+, a condition roughlycomparable to the physiological condition (Fig. S2). Theblocking effect of 10 μM SPM (with the physiologicalconcentration ranges in mammalian cells [9, 24, 65]) issimilar in different extracellular K+ concentrations whetherthe membrane potential is −40, +20, or +40 from the rever-sal potential (the intracellular K+ concentration was fixed at100 mM). These results lend a further support that thefindings in symmetrical 100 mM K+ may be extrapolatedto envisage the Kir2.1 channel behavior in more physiolog-ical conditions. These results are also consistent with a viewthat internal SPM block is flow-dependent and that thepermeating K+ may compete with SPM for the same sitesin the pore.

Dramatic decrease of IR index and emergence of a slowunbinding phase of internal SPM block in specific E224mutant channels

The E224G and E224Q mutations have been shown to affectthe inward rectifying block of the Kir2.1 channel by intra-cellular SPM [12, 25, 56, 57, 63]. E224 in the cytoplasmic

domain thus very likely plays a critical role in the making ofa pore conformation which is critical for SPM binding aswell as the flow-dependent SPM block of the Kir2.1 channel[4, 6, 11, 31, 73]. Figure 2a shows that the Kd values arehigher in the E224G and E224Q mutant channels than that inthe WT channel at every voltage. Moreover, the Kd–Vm plotno longer shows a dramatic change around the reversalpotential. The IR index in the mutant channels thus is muchsmaller than that in the WT channel (Fig. 2b). We also triedthe other substitutions (A, C, N, W, and R) for E224 and gottwo more mutant channels (E224A, E224C) showingenough currents for analysis. The findings are qualitativelysimilar among different E224 mutant channels (see alsoFig. 3d). Interestingly, there is a “slow tail” in the interven-ing −100 mV pulse in the mutant channels in the presenceof internal SPM, but not in the control condition (Fig. 2c).The slow tail thus is very likely ascribable to the slowunbinding of SPM from a pore-blocking site. Consistently,the slow tail shows the same decay time constantirrespective of the length at the +100-mV pulse. The am-plitude of the slow tail, however, tends to be larger withlengthening of the +100-mV pulse before reaching a satu-rating level. The development of the slow tail therefore maysignal the course of SPM binding to this slow unbindingsite at the preceding pulse at +100 mV and indeed shares avery similar time course of the decay of the macroscopicoutward currents at +100 mV (Fig. 2d). A closer look at thesaturating amplitude of slow tail in Fig. 2c, however, revealsthat it is always ∼20% (and the fast phase is always ∼80%) ofthe total inward current irrespective of different SPM concen-trations (Fig. 2e). This finding lends a further support to theidea that SPM block is flow dependent (and thus the SPMbinding site is located in a flux coupling pore segment).Specifically, it implies that SPMmay be pushed by the inwardor outward K+ flux to take a more internal or more externalposition in this segment and consequently has differentblocking effects on K+ flow (see “Discussion” and Fig. 10for details). Similar slow tails can also be found in the E224A,E224C (data not show), and especially the E224Q mutantchannel where the decay and the development of the slow tailare extremely slow (see Fig. 4a for sample sweeps).

Negligible voltage dependence in the binding kineticsbut strong voltage dependence in the unbinding kineticsof internal SPM

The binding rate of SPM to this flow-dependent blockingsite is barely voltage dependent (δ ∼0.04; Fig. 3a, b). Incontrast, the unbinding rate is strongly voltage dependent(δ ∼0.57 with the presumption of four effective charges onSPM, Fig. 3c). Figure 3d further shows that the kinetics of

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the development and those of the decay of the slow tail (i.e.,the on and off rates of SPM) are closely correlated witheach other in every different E224 mutant channels. Be-cause the voltage dependence of on and off rates is obtainedwith preponderant outward (+90∼+120 mV) and inward cur-rents (−50∼−100 mV), respectively, the foregoing voltagedependence should be essentially devoid of flux couplingeffect and thus provides a rather accurate measurement of thelocation of the SPM blocking site (see Fig. 10). These findingsaltogether indicate that E224 mutations induce a pore-narrowing change right at the internal end of the flux coupling

segment containing the SPM blocking site. The mutant chan-nels therefore not only have markedly decreased SPM affinity,but also show correlative slowing in both binding and unbind-ing kinetics of SPM.

Pore narrowing induced by E224 mutations at the bundlecrossing region of M2

Because E224 is located in the cytoplasmic domain, a verywide part of the pore [17, 55, 64], we then tried to define theactual location of the narrowed pore region in E224 mutant

Fig. 5 Similar voltagedependence of the SPMunblocking kinetics in the WTand E224G and M183W mutantKir2.1 channels. a The two-pulseprotocol is the same as that inFig. 2c except that the secondnegative pulse is set to −40 ratherthan −100 mV. Representativecurrent traces were recorded incontrol and in 100 μM internalSPM, demonstrating that theslow tail is also present in theWT Kir2.1 channels in thepresence of SPM at“appropriate” membranepotentials. b The inverses of therelaxation time constants of theslow tail currents are plottedagainst membrane potential insemilogarithmic scale for theWTand different (single as well ascorresponding double) mutantKir2.1 channels (n=5–8 for eachchannel). The two-pulse protocoland data analyses are the same asthat in Figs. 2c and 3c. Thesecond negative pulse in thesame two-pulse protocol was setto −40∼−100 mV to examine thevoltage dependence of SPMunblock from the binding site.The off rate(0) and Zδare ∼44 s−1 and 1.7 for the WTchannel, ∼19 s−1 and 1.6 for theM183W, and ∼1.0 s−1 and 1.9 forthe E224G/M183W mutantchannels, respectively. TheE224G mutant channel data(solid line) are taken from Fig. 3cfor comparison

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channels with concomitant mutations. Interestingly, the ex-tremely slow binding and unbinding processes of internalSPM in the E224Q mutant channel are dramatically acceler-ated by concomitant mutations at the bundle crossing regionA178C, A178T, and M183W (Fig. 4a). We also examinedthe decay of macroscopic outward currents at +100 mV inthe E224Q/A178C, E224Q/A178T, and E224Q/M183Wdouble mutant channels. The decay of outward currents at+100 mV once more shares a similar time course to thedevelopment of slow tails in these double mutant channels

(Fig. 4b, c.f. Fig. 2d). It has been shown that internal MTSreagents can modify WT Kir2.1 channel [39, 46], implyingthe existence of endogenous cysteine residues in the innerpart of the pore (e.g., residue C169 which is located externalto the bundle crossing region of the pore). We thereforeexamined the effect of A178T or M183W mutation on theMTSEA modification rate in the preexisting E224Q mutantchannel. Figure 4c shows that 100 μM internal MTSEAquickly modifies the WT, A178T, and M183W mutant chan-nels, but modifies the E224Q mutant channel with a much

Fig. 6 Inhibition of the E299mutant Kir2.1 currents byinternal SPM. a The Kd in theE299S and E299A mutantchannels is plotted againstvoltage (each points obtainedwith the same approach as that inFig. 1c, n=6–11). The IR indicesare ∼21 and ∼18 for the E299Sand E299A mutant channels,respectively. The WT channeldata (solid line) are taken fromFig. 1c for comparison. b Theslow tails are present in theE299S and E299A mutantchannels (at −100 mV) in100 μM SPM (see Fig. 2c for thepulse protocol). The dotted linesare monoexponential fits to thegrowing amplitude of theslow tail with time constantsof ∼7 and ∼20 ms for the E299Sand E299A mutant channels,respectively. c The average of thereverses of time constants frommonoexponential fit to thecourse of development ofthe slow tail in part c (on rateat +100 mV) is plotted againstthe average of the reverse of thedecay time constant of the slowtail itself (off rate at −100 mV).The dashed line is a linearregression demonstrating thelinear correlation between thetwo parameters in differentmutant channels (n=4–11, thesame approach as that in Fig. 3d)

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slower speed, demonstrating the existence of MTS-sensitiveelements beyond the narrowed region caused by E224Qmutation in the pore. Interestingly, MTSEA modificationrates are significantly accelerated in the E224Q/A178T andE224Q/M183W double mutant channels when comparedwith the E224Q (single) mutant channel. These findingsindicate that E224Q mutation changes the pore size at thebundle crossing region of M2 (178–183) to affect thebinding/unbinding process of SPM. In this regard, it is inter-esting to note that specific single mutations involving A178,M183, and A184 also decrease the IR index by 10 to morethan 1,000 times, consistently implicating the involvementof the bundle crossing region in the inward rectifying blockby internal SPM (Fig. 4d).

Similar voltage dependence in SPM unbinding kineticsin different mutant channels

Figure 5a shows that the slow tail ascribable to SPM unbind-ing is also discernible in the WT channel, although only at amuch less hyperpolarized intervening potential (i.e., −80 mVor more positive potentials). Figures 5b and S3 summarizedthe decay kinetics (the unblocking rates of SPM) in the WT,and in the single as well as double mutant channels involving

E224, and M183. The monotonous deceleration with mem-brane depolarization indicates slower off rates of the blockingSPM at more positive potentials. Although there is a ∼1,000-fold difference in the absolute values of the off rates, thevoltage dependence remains rather similar among the WTand different mutant channels, suggesting significantly alteredheight but not location of the major (rate limiting) barrier forthe blocking SPM to exit the pore (to the intracellular side).More detailed quantitative analyses of the decay kinetics inFig. 5b and the binding affinity of SPM in relevant single aswell as double mutant channels (Fig. S4) would lend furthersupport of the actual site of action in the bundle crossingregion for E224 mutations (see “Discussion”).

The same pore-narrowing effect of E299 mutationas that of E224

E299 is another residue previously reported to be closelyassociatedwith the function of Kir2.1 channel pore. Figure 6ashows that the IR indices are decreased to ∼21 and ∼18 in theE299S and E299A mutant channels, respectively. A slow tailsimilar to that in Fig. 2 is also found in the E299S and E299Amutant channels at −100 mV in the presence of 100 μMSPM. The slow tail again has the same decay time constant

Fig. 7 The strong cooperativeeffect of concomitant E224G andE299S mutations on internalSPM block. a Sample sweepswere recorded in 100∼300 μMinternal SPM in E224G/E299Sdouble mutant channel using thesame two-pulse protocol as thatin Fig. 2c. The dotted lines aremonoexponential fits to thegrowing amplitude of the slowtail with time constants of 350and 148 ms in 100 and 300 μMSPM, respectively. b The two-pulse protocol and data analysisare the same as that in Fig. 2c.The second negative pulse in thesame two-pulse protocol was setto −40∼−110 mV to examine thevoltage dependence of SPMunblock from the binding site.The off rate (0) and Zδ are∼0.1 s−1 and 1.7 for the E299Sand ∼3×10−3 s−1 and 2.6 for theE224G/E299S mutant channels,respectively. TheWTand E224Gmutant channel data (solid lines)are taken from Figs. 3c and 5bfor comparison

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irrespective of the length of the +100-mV pulse. The ampli-tude of the slow tail, however, tends to grow larger withlengthening of the +100-mV pulse until a plateau is reached.The development and the decay kinetics of the slow tails areonce more well correlated with each other in different E299mutant channels (Fig. 6b, c). These features are all verysimilar to the findings in the E224 mutant channels inFig. 2. We therefore investigated the possible interactionbetween E224 and E299 residue in more detail. The devel-opment of slow tails is evidently faster in 300 μM than in100 μM SPM (Fig. 7a), whereas the kinetics of slow taildecay are apparently unrelated to SPM concentration in theE224G/E299S double mutant channel (9.4±0.9 ms from fourpatches in 100 μMSPM and 9.6±0.1 ms from three patches in300 μM SPM; data not shown). Figure 7b shows the voltagedependence of the unblocking rates of SPM from the bindingsite in WT, E224G, E299S single mutant, and E224G/E299Sdouble mutant channels. The monotonous deceleration withmembrane depolarization indicates a slower (inwardly) offrate of SPM at more positive potentials. Moreover, the voltagedependence is again similar in the WT and the mutant chan-nels. These findings altogether again implicate that E224 andE299 mutations both have a very significant allosteric effecton the same pore element to make a narrowed segment in theconduction pathway (see “Discussion”).

The slow tail of SPM unblock is present in pHi 6.4,but not 8.4 in the E299H mutant channel at −100 mV

To investigate the functional role of the negative charge inE224 or E299, we examined the blocking effect of SPM withhistidine substitution of these residues. The macroscopic cur-rents of E224H mutant were too small to be analyzed precise-ly. On the other hand, we could document the time course ofdevelopment of internal SPM binding and unbinding to theE299H mutant channel at +100 mV in different internal pH(pHi) (Fig. 8a–c). We found that slow tail currents causedby internal SPM block are discernible in pHi 6.4 and 7.4,but not 8.4. The pattern of development of the slow tail inpHi 6.4 is very similar to that observed in E299S mutantchannels in Fig. 6b. However, the development and decayof the slow tails currents are evidently faster in pHi 7.4 thanin pHi 6.4. Of note, the blocking effect of the internal SPMon theWTchannel is very similar in pHi 6.4, 7.4, and 8.4 (interms of either the relative current inhibited by the internalSPM or the decay time constants of the slow tails; Fig. 8d–f). It is thus unlikely that the different findings on the slowtails in the E299H mutant channel in pHi 6.4∼8.4 areascribable to different protonation on the SPM moleculeitself at different pH. We conclude that the negative chargeat E299 very likely plays an essential role in the mainte-nance of the “normal” structure of the critical flux couplingpore segment containing the SPM blocking site.

Elimination of the slow tail in the E224 and E299 mutantchannels by concomitant mutations of R228 and R260

If E224 and E299 both control the conformation of a criticallong pore region responsible for the inward rectifying SPMblocking effect in Kir2.1 channels with their negativecharges playing an essential role in this regulatory process,then it would be desirable to explore the possible countercharges interacting with E224 and E299. We have seen anultraslow tail for SPM unblock in the E224Q mutant chan-nel. Interestingly, these ultraslow binding and unbindingprocesses of internal SPM are completely eliminated byconcomitant R228 or R260 mutations, which also acceler-ates SPM binding onto the channel already bearing theE299A mutation (Fig. 9a, b). In the meanwhile, the IRindex is partly recovered in these double mutant channelsin comparison with the E224Q, E299A, and E299S singlemutant channels (Fig. 9c). Also, the Kd of SPM is increasedsignificantly in the positive voltage range after the singlemutation of R228 or R260 (Fig. S5). These data altogetherindicate that E224 and E299 are responsible for keeping theflux coupling segment of the Kir2.1 channel pore “open,”most likely via an allosteric effect closely related to theelectrostatic interactions with specific positively chargedresidues (e.g., R228, R260) in the pore.

�Fig. 8 The effect of pHi on the slow tail in the E299Hmutant channel. aTheslow tail of SPM unblock is present when pHi is 6.4 and 7.4, but is absentwhen the pHi is adjusted to 8.4 in the E299H mutant channel (with the sameexperimental protocols as that in Fig. 2c). In pHi=6.4, the development ofslow tail in the E299Hmutant channel shares a very similar pattern to E299Sor E299A. bCumulative results are obtained for the experiments described inpart a. The decay time constants of the slow tail currents at −100 mV in theE299H channel are 1.7±0.1 ms (n=5) and 0.6±0.06 ms (n=5) for pHi 6.4and 7.4, respectively. There is no discernible slow tails in pHi 8.4. c Cumu-lative results are obtained for the experiments described in part a. The timeconstants of the developmental of the slow tail currents in the E299H channelat +100 mVare 11.4±0.1 ms (n=5) and 0.8±0.04 ms (n=5) for pHi 6.4 and7.4, respectively.dA two-pulse protocolwas used tomeasure the binding andunbinding kinetics of internal SPM. The patch was held at 0 mVand steppedtwice to −100 and −40 mV, respectively (20 ms each), with gradual length-ening of the intervening +100 mV period in the presence of 100 μM internalSPM for pHi 6.4 and 8.4 in theWTKir2.1 channels. The development of theslow tail in the second −40mV pulse is used as ameasure of SPM binding tothe flow-dependent blocking site at +100 mV. Part of the current sweeps inthe second −40 mV pulse is enlarged to demonstrate the very similar decaykinetics of the slow tails in both pHi. eCumulative results are obtained for theexperiments described in part d. The time constants of decay of the slow tailcurrents in the WT channel at −40 mVare 1.0±0.1 ms (n=4), 0.9±0.01 ms(n=5), and 0.9±0.1 ms (n=4) in pHi 6.4, 7.4, and 8.4, respectively. fCumulative results are obtained from five patches with the same pulseprotocol described in Fig. 1a in the WT channel. The relative currents aredefined by the ratio between the steady-state current amplitude in 100 μMSPM and that in control and are very similar in pHi 6.4, 7.4, and 8.4 over awide range of membrane voltages

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Discussion

The SPM binding site is located in a flux coupling segmentof the pore

We show that the Kd of SPM exhibits manifest flow depen-dence as there is always a significant change around the reversal

potential (Figs. 1c–e, S1, and S2). Also, the Kd of SPM isincreased considerably in both the positive and the negativevoltage ranges with a markedly decreased IR index in theD172N mutant channel (Fig. S6). In contrast, the Kd of SPMsignificantly increases chiefly in the positive voltage range withonly a moderate decrease in IR index in the S165L mutantchannel. The flux coupling segment containing the SPM

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blocking site thus seems to involve D172 and is demarcatedexternally by a large energy barrier closely related to S165 forSPM to transverse (Fig. 10e). The inner end of the segment, onthe other hand, seems to correspond to the inner end of thebundle crossing region (roughly M183–A184) (Figs. 4, 5, andS4). SPM would move in the flux coupling segment to take amore internal or more external position according to thedirecting of pore preponderant K+ flux. There is thus a differentblocking effect with the same concentration of SPM in differentdirections of K+ flux and two (one fast and one slow) phases inthe development of the inward currents upon membrane hyper-polarization following a depolarizing pulse in the presence ofinternal SPM (e.g., Fig. 2c; see also Fig. 10e).

The flux coupling segment is separated from the inside withan electrical distance ∼0.5 and a very asymmetrical energybarrier for SPM permeation

In previous reports, the measurement of the electrical dis-tance of the SPM blocking site could be contaminated by theflux dependence of the block and probably also by theoutward exit of the blocking SPM especially at positivepotentials. This may be part of the reason why there aresignificant discrepancies among the electrical distance ofthe SPM blocking site in different reports [12, 24, 25, 41,49]. We have endeavored to minimize the contamination offlux-dependent effect by dissecting the dissociation constant,a steady-state parameter, into the binding (on) rate and un-binding (off) rate of the blocking SPM and measured thebinding and unbinding kinetics with appropriately prepon-derant direction of the K+ flux (i.e., outward and inwardpreponderant flux, respectively). This approach not onlygives an electrical distance of the SPM binding site (δ∼0.5) relatively devoid of the contamination from flow de-pendence, but also reveals a very asymmetrical barrier forinternal SPM to traverse before it reaches the blocking site inthe pore (Fig. 10). Based on the binding/unbinding rateconstant at 0 mV [33, 72] and Erying rate theory [20], wehave constructed an energy profile of SPM in the Kir2.1channel pore (Fig. 10e). We would propose that E224 andE299 are allosterically involved in both the binding/unbindingprocesses of internal SPM to/from the blocking site and theflux coupling properties of the Kir2.1 channel pore. This rela-tively small changes in Zδ of the unbinding rates of SPMbetween ∼1.5 and ∼2.6 in different mutant channels, in com-parison with the very large changes in barrier height (e.g., theunbinding rates of SPM in the E224G/E299S double mutantchannel is slowed by ∼1,000 times than in the WT channel,Fig. 7), would signal the bioenergetic consistency of the posi-tion of this important barrier in the Kir2.1 pore. The molecularsubstrate of this very asymmetrical energy barrier peak at δ ∼0may involve, for example, a more abrupt reorientation of thepermeating cations and/or the coordinating ligands at the

internal end of the flux coupling pore segment. The require-ment of orientation of the permeating cation may be part of thereason why the more asymmetrical molecules such as SPMshall face a higher barrier at this point than K+.

The bundle crossing region constitutes the flux couplingsegment critical for SPM block of the Kir2.1 channel pore

We have noted that point mutations involving E224 and E299could largely or even completely abolish the inward rectifyingfeatures of SPM block (Figs. 2 and 6). Quantitative analysesof these off rates and the Kd changes in double mutantchannels (Figs. 6 and S4) further support the idea that themutations involving E224 and the bundle crossing region(e.g., M183) actually have an effect on the same locus of thepore. For example, E224G and M183N single mutations slowdown the unbinding of SPM by 60 to 150 times (based on thedata at −70 mV), whereas there is a ∼830-fold slowing inunbinding kinetics observed in the E224G/M183N doublemutant channel (Fig. S3). Kinetically speaking, if the E224Gor M183N single mutation increases the height of differentbarriers to make the SPM unbinding rates 60- to 150-foldslower than in the WT channel, then the E224G/M183Ndouble mutation should slow the unbinding by no morethan ∼210 (e.g., 60+150) times. The observed ∼830-foldslowing in the double mutant channel thus would stronglyindicate that the two mutations actually act on the samepermeation barrier. Moreover, M183N and M183W singlemutations have a very similar effect to E224G mutation onthe binding/unbinding kinetics of SPMand/or IR index (Figs. 5and S3). Also, concomitant A178C, A178T, and M183Wmutations could greatly accelerate the very slow SPM unbind-ing rate in the presence of E224Q mutation (Fig. 4). The veryslowmodification rates ofMTSEA associated with the E224Qsingle mutation are also speeded up considerably by concom-itant mutations A178T or M183W (Fig. 4). These findings allindicate that what the imperative interactions among the resi-dues in the cytoplasmic domain aim to keep is the conforma-tion of the pore at the bundle crossing region. This bundlecrossing region thus may constitute another key structure inaddition to the selectivity filter in the Kir2.1 channel pore, andthe side chains of A184 and M183 of the bundle crossing poreregion are probably located in the narrowest pore regioninternal to the selectivity filter (Fig. 10d). Unlike the selectivityfilter which is located at the external pore mouth and respon-sible for the K+ ion selectivity of the pore, the bundle crossingregion is located near the internal end of the central cavity andseems to bear several sequentially arranged ionic sites to makethe characteristic “extrinsic” inward rectification of the pore.The extrinsic blocker SPM thus has to cross the peak of anasymmetrical energy barrier at the inner end of the bundlecrossing region to enter or to exit the flux coupling segment(and get back to the cytoplasmic pore domain). This bundle

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crossing region may also be responsible for the “intrinsic”rectification of the Kir2.1 channel because it is involved inthe buildup of a very asymmetrical barrier. Whether there is amanifest intrinsic rectification would be dependent on theheight of the barrier for the permeating ions (i.e., the barrier'srole as a rate-limiting step in the whole permeating process fora particular ion).

The pore caliber can be so minimized at the bundle crossingregion as to markedly slow the movement of SPM

The markedly and concomitantly slowed binding and unbind-ing rates of the blocking SPMwith mutations involving E224,E299, and the bundle crossing region indicate a narrowing of

this segment of the pore (e.g., Figs. 3, 4, 5 and 6). The reducedMTSEAmodification rates with E224Q, A178T, andM183Wmutations further support this idea (Fig. 4). In some cases, thenarrowing may be so marked that even the movement of K+

ion across this region becomes the rate-limiting step of thewhole permeation process and thus results in the reducedsingle channel conductance [5, 12]. The reduced single-channel conductance and the increased open-channel noisein E224G and Q and K mutant channels would then signalelevation of the energy barrier [5, 39] and a destabilizedconformation of the bundle crossing region. The narrowestpart of this region (the local free energy peak for ion perme-ation) should be very close to or essentially “face” the intra-cellular solution in terms of electric distance (δ=∼0) (Fig. 10e).

Fig. 9 Concomitant mutations of the R228 and R260 residues elimi-nate the slow component of SPM unblock in E224 and E299 mutantchannels. a Sample sweeps demonstrating inhibition of E224Q/R260Nand E224Q/R228Q double mutant channels by 300 μM internal SPM insymmetrical 100 mM K+. The two-pulse protocol is the same as that inFig. 2c. Note that 300 μM internal SPM blocks these double mutantchannels with a very fast speed as evidenced by the rapid decrease of theoutward currents upon membrane depolarization. Accordingly, the slowtails which are very much manifested in the E224Q single mutantchannel at the following hyperpolarizing pulse (Fig. 4a) are no longerdiscernible in these double mutant channels. b Cumulative results are

obtained from five patches with the same protocol describe in part a forthe E224Q, E299A, E224Q/R228Q, E224Q/R260N, and E299A/R228R mutant channels. The tauon, 1 and tauon, 2 denote the timeconstant of the growing course of the slow tails and the time constantof the decay of the macroscopic outward currents in 300 μMSPM. Notethat the tauon, 1 is not available in the E224Q/R228Q, E224Q/R260N,and E299A/R228R mutant channels because of the lack of discernibleslow tails. c The IR index is partly recovered in the E299A/R228A,E224Q/R260N, and E224Q/R228Q double mutant channels in compar-ison with the E224Q or E299A single mutant channels

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From this narrowest part outward, there are functionallycoupled and thus very likely structurally contiguous ionicsites elongating for an electrical distance of ∼0.5 to lead tothe final SPM binding position in the central cavity (∼D172or just before S165). It is intriguing that a stretch of roughly14 amino acid residues from K185 to D172 would make themultiple ionic sites responsible for flux coupling, especiallyin view that many of these residues are hydrophobic ones.The possibility of making an ionic or SPM binding site withside chains from residues in different subunit thus should beconsidered. In any case, the mutations causing narrowing ofthe pore are in general the same as those interfering with theinward rectification feature of the pore, suggesting thatdisruptions of the coupling between different ionic sites orof the sites themselves are consequences of relatively majorrather than delicate conformational changes. The bundlecrossing region, or the junction between the central cavityand the intracellular domain, of the Kir2.1 channel porethus seems to have the potential of undergoing some majorconformational changes. This is consistent with the crys-tallographic data of the Kir channel family [7, 18, 23, 52,64]. These conformational changes very likely involvewidening and narrowing of the pore, well simulating open-ing and closing of a channel gate. This potential gatingmovement, however, seems to be greatly (but may not becompletely) sacrificed in the WT channel for a more fixedstructure to assure ideal coupling among the ionic sites inthis region.

E224/E299 allosterically controls the conformation of thebundle crossing region via electrostatic interactions withR228/R260

According to the structural data of Kir2.1 and Kir2.2 chan-nels [18, 55, 64] and calculations based on the foregoingstructural templates using DS v2.5 software (see “Materialsand methods”), E224 and E299 are located in the widenedcytoplasmic domain [18, 55, 64], which is probably toowide to make the flux coupling permeation feature. Wehave further demonstrated that E224G and E299S muta-tions slow down the unbinding of SPM by 60–30 times,whereas there is a ∼950-fold slowing in unbinding kineticsobserved in the E224G/E299S double mutant channel(Fig. 7). Analogous to the quantitative analysis we madefor the E224G and M183N single mutant channels and theE224G/M183N double mutant channels in Fig. S3, this is astrong evidence for the same site of action for the E224Gand E299S mutations in terms of slowing SPM unbinding.The apparently unchanged voltage dependence of the un-binding kinetics for E224G and E299S single mutant aswell as E224G/E299S double mutant channels is also con-sistent with a common site of action in the flux couplingsegment by E224G and E299S mutations (Fig. 7). In other

words, based on similar quantitative argument, theA178, M183, E224, and E299 mutations all seem tohave their major effect on the same locus of the channel

�Fig. 10 Schematic drawing illustrating the long pore region of the Kir2.1channel. aThe homologymodel is constructed based on the correlativeX-ray crystal structures of the mouse Kir2.1 and chicken Kir2.2 channel (see“Materials and methods”). Four subunits of the homology modeling ofKir2.1 channel are shown. b A regional view of the cytoplasmic domainin the homology model of Kir2.1 tetramer from the intracellular side. c Adiagram of two subunits of the Kir2.1 cytoplasmic domain. The sidechains of E224, R228, R260, and E299 are indicated with sticks ofdifferent colors. The enlarged pore portion demonstrates that the predictedinter-residue distances (from side chain tip to tip) between E224 andR228, E224 and R260, and E299 and R228 are ∼17, ∼10, and ∼5 Å,respectively. d Side view of the Kir2.1 channel showing the transmem-brane domain of the two subunits. The transmembrane domain is coloredgray. The side chains of M183 and A184 are indicated with sticks ofdifferent colors. In the narrowed pore portion, the predicted inter-residuediagonal distances (from side chain tip to tip) between residues of M183and A184 are ∼18 and ∼11 Å, respectively. e A drawing of the energyprofile of SPM in the pore of the Kir2.1 channel. The flux couplingsegment of the Kir2.1 channel pore probably involves D172 as its mostexternal ionic site and is demarcated externally by a large energy barrierroughly at S165 for SPM to cross (i.e., surmountable only at very positivepotential). Because the exact location of this barrier is not investigated inthis study, it is drawn in dotted lineswithout a definite peak to indicate theuncertainty. The binding kinetics of SPM is essentially voltage indepen-dent (Fig. 2), whereas the unbinding rates of SPM (the kinetics of thedecay of the slow tails) show a Zδ value of 1.5∼2.6 in theWTchannel andmany different mutant channels (Fig. 5). Assuming a Z value of 4 forSPM, we shall then locate D172 at δ∼0.5 from binding site. The majorenergy barrier therefore should have a peak located right at δ∼0 and thenslopes down to the SPM blocking site at δ∼0.5 based on the voltagedependence of the binding and unbinding kinetics. However, there wouldbe no other comparable deterring mechanism all the way to D172 onceinternal SPM pass this initial barrier. This is consistent with the biophys-ical basis of a flux coupling segment responsible for a prominent long-pore effect, namely contiguous ionic sites separated by insignificantenergy barriers. In other words, the segment between the energy barrierpeak and the well should contain shallow ionic sites common for the K+

and SPM, but we have omitted those details in the drawing for the sake ofsimplicity. The blocking SPM in this flux coupling segment thus can takedifferent positions according to the K+ flux. When there are outward K+

currents (preponderant K+ efflux in the segment), SPM would be pushedto the most external position, right against another large energy barrieraround S165 in a relatively small caliber part of the flux coupling segmentadjacent to the selective filter. The outward K+ currents thus would be(nearly) completely blocked. When there are inward K+ currents (prepon-derant K+ influx in the segment), SPM would tend to take the innermostposition. Here is probably the relatively wide part of the segment and thusless pronounced block of the K+ current flow by SPM. The blocking SPMat this innermost position would need to cross the energy barrier at theinner end of the bundle crossing region to exit the flux coupling segmentand thus the pore is completely unblocked. This is why there is an initialfast phase followed by a slow phase (slow tail) in the development of theinward currents uponmembrane hyperpolarization following a depolarizingpulse in the presence of the internal SPM (e.g., Fig. 2c). The inner part ofthis flux coupling segment corresponds to the bundle crossing area of M2and is capable of certain apparent open-closing conformational changes.E224 and E299 in the cytoplasmic domain, possibly via interactions withR228 andR260, play an imperative role in keeping the open and appropriateconformations of this bundle crossing region via allosteric effects

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pore to slow SPM unbinding (Figs. 5b, 7b, and S3).Moreover, the extent of a positively charged side chainat position 299 (protonation of E299H) could be wellcorrelated with the pore narrowing and slowing theSPM unbinding (Fig. 8). Also, the effect of E224Q andE299A on IR index and the inward exit kinetics of SPM is

reversed by concomitant mutations of positive-charge resi-dues R228 and R260 (Fig. 9), and the distance betweenthe charges in E224 and R260 or in E299 and R228 isprobably only 5–10 Å (Fig. 10c). It is plausible thatelectrostatic interactions in the cluster of E224, E299,R228, and R260 play a critical role in assuring a stable

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and open conformation of the flux coupling or bundlecrossing pore region responsible for coupled ion movement(see also [5, 6, 12]).

Acknowledgments This work was supported by National Health Re-search Institutes, Taiwan, grants NHRI-EX101-10006NI and NHRI-EX102-10006NI (to C.-C.K.), and also by National Science Council,Taiwan, grant NSC95-2320-B-002-064-MY3 (to C.-C.K.).

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