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Cellular and Molecular Neurobiology, Vol. 24, No. 3, June 2004 ( C© 2004)

Intramembrane Charge Movement Associated WithEndogenous K+ Channel Activity in HEK-293 Cells

Guillermo Avila,1 Alejandro Sandoval,2 and Ricardo Felix2,3

Received May 9, 2003; accepted September 19, 2003

SUMMARY

1. The use of molecular biology in combination with electrophysiology in the HEK-293cell line has given fascinating insights into neuronal ion channel function. Nevertheless, tofully understand the properties of channels exogenously expressed in these cells, a detailedevaluation of endogenous channels is indispensable.

2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+,and Cl− channels and this predicts that changes in membrane potential will cause intramem-brane charge movement, though this gating charge translocation remain undefined. Here,we confirm this prediction by performing patch-clamp experiments to record ionic and gat-ing currents. Our data show that HEK-293 cells express at least two types of K+-selectiveendogenous channels which sustain the majority of the ionic current, and exclude a signifi-cant contribution from Ca2+ and Cl− channels to the whole-cell current.

3. Gating currents were unambiguously resolved after ionic current blockade enablingthis first report of intramembrane charge movement in HEK-293 cells arising entirely fromendogenous K+ channel activity, and providing valuable information concerning the acti-vation mechanism of voltage-gated K+ channels in these cells.

KEY WORDS: K+ channels; Ca2+ channels; gating currents; HEK-293 cells.

INTRODUCTION

The human embryonic kidney (HEK-293) cell line provides a frequently used ex-pression system for investigating ion transport systems following transfection of theappropriate cDNA from a variety of tissues including the nervous system. Duringsuch studies on recombinant neuronal voltage-gated ion channels the presence ofendogenous conductances has been noted. Since the occurrence of these voltage-dependent conductances has implications for the study of exogenously expressedion channels and in fact any protein implicated in ion transport through the plasma

1 Department of Biochemistry, Center for Research and Advanced Studies of the National PolytechnicInstitute (Cinvestav-IPN), Mexico City, Mexico.

2 Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studiesof the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico.

3 To whom correspondence should be addressed at Departamento de Fisiologıa, Biofısica y Neurocien-cias, Cinvestav-IPN, Avenida IPN 2508, Colonia Zacatenco, Mexico D.F., CP 07300, Mexico; e-mail:rfelix@fisio.cinvestav.mx.

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membrane, we went on to characterize the biophysical properties of these conduc-tances further.

Previous studies have shown that endogenous voltage-gated Ca2+, Cl−, and K+

channels exist in HEK-293 cells (Berjukow et al., 1996; Jiang et al., 2002; Yu et al.,1998; Zhu et al., 1998). It has been unclear, however, what is the contribution ofthe different types of endogenous voltage-gated channels to the whole-cell current,and more importantly a complete characterization of the currents observed is lack-ing. In particular, the charge movement associated with ion channel activation hasnot been previously documented in HEK-293 cells. Intramembrane charge move-ment is the result of multiple conformational changes involving positively charged,transmembrane regions that function as the voltage sensors for gates that controlchannel opening and closing (Larsson, 2002). Displacement of these charge-bearingintramolecular domains, in response to changes in the transmembrane electric field,produces gating currents (Armstrong and Bezanilla, 1973).

The data presented here extends the investigation on the endogenous currentsin the HEK-293 cell line by examining the contribution of the distinct voltage-gatedion conductances to the whole-cell current, and are the first to indicate that thesecells display a strong nonlinear intramembrane charge movement. In addition, herewe show that virtually all this charge movement is related to the robust activity ofendogenous K+-selective channels, with little or no contribution from voltage-gatedCa2+ and Cl− channels. Gating current analysis provided valuable information aboutthe activation mechanism of the endogenous K+ channels in HEK-293 cells because itreflected transitions that have not been observed in previous ionic current recordings.

MATERIALS AND METHODS

Cell Culture

Human embryonic kidney (HEK-293 cells) were grown in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% equine serum, 2 mM L-glutamine,110 mg/L sodium pyruvate, and 50 µg/mL gentamycin, at 37◦C in a 5% CO2–95%air humidified atmosphere. Cells were fed every other day, subcultured once weekly,and seeded at a density of ∼105 cells/cm2 in poly-L-lysine-coated coverslips for elec-trophysiological recording.

Electrophysiology

Cells were subject to voltage-clamp experiments under the whole-cell variantof the patch-clamp technique (Marty and Neher, 1995) following 3–4 days of initialplanting. All experiments were carried out at room temperature (∼22◦C) and theholding potential (HP) was−80 mV. Currents were recorded with an Axopatch 200Bamplifier and filtered at 2 kHz (four-pole Bessel filter). Ion and gating currents (seebelow) were digitized at 10 and 200 kHz, respectively using a DigiData 1200 interfaceand analyzed using the pCLAMP and SigmaPlot software suites. Linear capacitativecurrents were minimized analogically using the capacitative transient cancellationfeature of the amplifier. The remaining linear components were subtracted using a

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Table I. Recording Solutions

Solution K-Asp Cs-Asp NaCl TEA-Cl KCl CaCl2 MgCl2 Glucose CdCl2 LaCl3 HEPES EGTA

A 140 3 5 5 10B 140 3 5 5 10C 145 10 10D 145 10 0.5 0.3 10E 130 8 0.2 5 10 10F 120 5 10 10

Note. Units are in mM. The pH was adjusted to 7.3 with NaOH (A), TEAOH (B, C, and D), KOH (E),or CsOH (F).

P/8 protocol. Membrane capacitance (Cm) was determined as described previously(Meza et al., 1994) and used to normalize gating currents (nC/µF) obtained fromdifferent cells (Avila and Dirksen, 2000). Briefly, the Cm values were determinedby applying a series of three consecutive pulses (20 ms; −20 mV) from the HP, andintegrating the current traces that resulted from subtracting the capacitive transientsassociated to the patch pipette (cell-attached conditions) from the total capacitivetransients obtained immediately after breaking into the cell (whole-cell conditions).The recording solutions are given in Table I. Patch pipettes were made from borosil-icate glass and the typical electrical resistance was 4 MÄ when filled with internalsolutions (E and F; Table I).

Ion and Gating Currents

Macroscopic ion currents were acquired in response to 200 ms test pulses ofvariable amplitude at a sampling frequency of 10 kHz. Current to voltage relation-ships (I–V curves) were obtained by plotting the peak amplitude of ion currents as afunction of their respective membrane potential during the test pulse. For properlyresolving gating currents, we increased the sampling frequency to 200 kHz and used5 ms test pulses of variable amplitude. To obtain the voltage dependence of chargemovement (Q–V curves), the nonlinear capacitative currents recorded at the onset“on” and the “off” of the test pulses were integrated and the resulting intramembranecharge movements (“Qon” and “Qoff”) were plotted as a function of the membranepotential during the test pulse. Subsequently, the Q–V curves were fitted according toa Boltzmann equation of the form, Qx = Qmax/(1+ exp[(V1/2 − Vm)/k]), where Qx

is either Qon or Qoff, Qmax is the maximum intramembrane charge movement, V1/2

is the voltage for half activation of Qmax, Vm is the membrane potential during thetest pulse, and k is a slope factor. All data are presented as mean ± SEM. Statisticaldifferences were determined to be significant at the p < 0.05 value, unless specified(unpaired t test).

RESULTS

K+ Channels Represent the Majority of Functional Ion Channels in HEK-293 Cells

We first aimed to record endogenous macroscopic voltage-gated ion currents inHEK-293 cells, under control conditions (Fig. 1). Toward this end, we carried out

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Fig. 1. TEA markedly decreases the amplitude of endogenous outward currents in HEK-293 cells.(A) Ion currents recorded in standard or control solutions (Na+/K+, left) and in the presence of 140 mMextracellular TEA (TEA/K+, right). The internal solution E was used for both experiments, whereasthe external solutions A and B were used for the Na+/K+ (left) and TEA/K+ (right) experiments,respectively (see Table I). The current traces were elicited from an HP of −80 mV, in response to testpulses of 200 ms to the indicated membrane potentials (Vm, mV). (B) Average peak current to voltagerelationships for ion currents recorded as in A (Na+/K+, solid circles; TEA/K+, open circles). Theaverage values for the cell membrane capacitance (Cm) are being plotted for each condition (inset). Foreach experimental condition, n = 6.

voltage-clamp experiments using standard recording solutions, which mainly con-sisted of 140 mM NaCl, 3 mM KCl, 5 mM CaCl2 (external solution A; Table I),130 mM K-Asp, and 8 mM KCl (internal solution E; see Table I). Under these con-ditions, the total whole-cell current mostly consisted of an outward current, whoseamplitude increases progressively with membrane depolarization with an apparentthreshold for activation at ∼0 mV (Fig. 1(A) and (B)). This current was hypoth-esized to represent a net efflux of K+ and/or a net influx of Cl− ions, as its am-plitude increased in proportion to these ions driving force. It has been reportedthat HEK-293 cells endogenously express TEA-sensitive K+ (Jiang et al., 2002;Yu and Kerchner, 1998) and voltage-gated Cl− channels (Zhu et al., 1998). Thus,we decided to estimate the specific contribution of these channels to the outwardcurrent in our batches of cells. Figure 1(A) shows representative current traces thatwere recorded following substitution of extracellular Na+ for TEA (i.e. by usingthe external solution B instead of A; Table I). As can be seen, the outward cur-rents generated in the presence of TEA are smaller and tend to activate at morepositive potentials. On average, the TEA significantly reduced the outward currentamplitude to ∼20% of its control value at +90 mV, and shifted its threshold for ac-tivation by ∼+40 mV (Fig. 1(B)). This reduction cannot be explained by possiblecell-to-cell differences in size given that the average membrane capacitance (Cm)values were practically the same for both experimental conditions (Fig. 1(B), in-set). Together, these results strongly suggest that nearly 80% of the macroscopic ioncurrent in HEK-293 cells is being carried through voltage-gated TEA-sensitive K+

channels.As for the TEA-insensitive ion current, it could still be carried by K+ efflux

and/or Cl− influx. To distinguish between these two possibilities, we compared ioncurrents recorded in the presence of extracellular TEA (as in Fig. 1, TEA/K+) with

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Fig. 2. Internal Cs+ but not niflumic acid eliminates the TEA-resistant ion current. (A) Ion currentsrecorded as in Fig. 1, in the presence of either extracellular TEA (solution B, left) plus 400 µM ofniflumic acid or extracellular TEA without niflumic acid (solution C, right). The main monovalention in the internal solution was either K+ (solution E, TEA + niflumic/K+, left) or Cs+ (solution F,TEA/Cs+, right). (B) Average peak current to voltage relationships for current traces recorded as inA (TEA+ niflumic/K+, solid squares; TEA/Cs+, open triangles). The inset represents the calculatedvalues for cell membrane capacitance (Cm). In both cases, the standard error (SEM) is smaller thanthe size of the symbols. Data represent the average from a total of three (TEA + niflumic/K+)and nine (TEA/Cs+) cells. Experimental data from Fig. 1(B) for the TEA/K+ condition are beingreplotted as a comparison (open circles).

ion currents that were acquired in the presence of extracellular TEA but substitutingK+ for Ca2+ and Cs+ in the external and internal solutions, respectively (solutionsC and F; Table I). Interestingly, the substitution of K+ from the recording solutionsfully eliminated the TEA-insensitive outward current at all voltages tested (Fig. 2(A),TEA/Cs+). Thus under these experimental conditions we were practically unableto detect any significant voltage-dependent ion currents (Fig. 2(A) and (B)). Theseresults indicate that virtually all of the endogenous ion currents of HEK-293 cellsare being carried by K+ ions. In support to this interpretation, we were unable tosee any reduction in current amplitude in the presence of the external solution Bsupplemented with niflumic acid (400 µM), a pharmacological agent that preferen-tially blocks Cl− channels (Scott et al., 1995) (Fig. 2(A) and (B)). Taken together, ourdata strongly suggest that HEK-293 cells do not express significant levels of func-tional voltage-gated Cl− channels, and that K+ channels are the main functional ionchannels in these cells.

Interestingly, as can be seen in Figs. 1 and 2, the activation threshold for theTEA-insensitive component of the whole-cell K+ current endogenously expressedin the HEK-293 cells is shifted several tens of mV to more positive potentials. Thisobservation is important since it may provide relevant clues regarding the identity ofthis component of the current. Hence, to evaluate more accurately the activation ofthe expressed currents, conductance–voltage (G–V) relationships were constructed(Fig. 3). To this end, the conductance (GK) at each membrane potential was esti-mated as GK = IK/(Vm − VK), where IK was peak current amplitude and Vm − VK

represented the driving force. Subsequently, the resulting G–V curves were fittedto the Boltzmann equation (see Materials and Methods section). This analysis re-vealed a shift of ∼40 mV in the G–V relationship for the TEA-insensitive current

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Fig. 3. Activation curves for endogenously expressed K+ channels in HEK-293 cells. (A)Conductance–voltage (G–V) relationships for ion currents from Fig. 1. The G–V curves weregenerated from peak currents after activating steps to distinct voltages as listed. The conduc-tance values were estimated as detailed in Results section, and the resulting G–V curves fromeach experiment were fitted to the Boltzmann equation. Average maximal conductance (Gmax),slope factor (k), and membrane potential to obtain a 50% of Gmax (V1/2) were 3.5± 0.6 nS, 21.2±2.6 mV, 37.8± 5.9 mV; and 1.3± 0.5 nS, 18.1± 5.2 mV, 77.5± 7.0 mV, for the TEA-sensitive (solidcircles) and insensitive (open circles) components of the whole-cell K+ current, respectively. Thesolid curves were generated using these average values. Gmax and V1/2 are significantly differentbetween the two different experimental conditions (p < 0.05). (B) Normalized G–V curves forexpressed K+ currents in HEK-293 cells. The data represented by the dashed lines were takendirectly from panel (A) and normalized to the corresponding Gmax values.

and showed that its activation was confined to positive potentials. In addition, theslope factor for this current was relatively high (18.1) indicating a weak voltage-dependence. These results strongly suggest the presence of an endogenously ex-pressed Ca2+-dependent K+ current in HEK-293 cells characterized by a very lowsensitivity to TEA. Beyond the scope of the current report, it may be questionedwhich Ca2+-dependent K+ channel(s) constitute(s) the native TEA-insensitive K+

current expressed in HEK-293 cells. Likewise, an interesting question arises as tohow these channels are activated in the absence of voltage-gated Ca2+ channels inthe plasma membrane of the cells.

Analysis of Endogenous Intramembrane Charge Movement in HEK-293 Cells

As can be seen in Figs. 1 and 2, most of the current traces exhibit nonlinearcapacitative transients that were virtually impossible to eliminate by combining theanalogical cancellation feature of the amplifier with an on-line subtraction protocol.We then surmised that these transients represented gating currents associated to theendogenous K+ channels of HEK-293 cells. Thus, we decided to investigate if thesecells were indeed expressing significant levels of endogenous gating currents. The cur-rent trace in Fig. 4(A) was obtained with the standard 200 ms protocol that we used torecord ion currents. This trace was digitized at a sampling frequency of 10 kHz. Evenat this sampling frequency, it was possible to observe nonlinear capacitative currentsat the onset “on” and the “off” of the test pulse. An interesting observation was that

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Fig. 4. Nonlinear capacitative currents recorded in the presence of extracellular TEA(solution C) and intracellular Cs+ (solution F). Current traces were elicited in responseto 200 ms (A, top left) or 5 ms (B, top right) test pulses to +30 mV, and digitized every100 µs (10 kHz) or 5 µs (200 kHz), respectively. Two milliseconds of each current traceare being expanded for clarity (bottom). The vertical scale in A applies to all traces.

both transients exhibit similar amplitudes, in response to a relatively strong depo-larization (+30 mV), suggesting that they may truly reflect intramembrane chargemovements, which are being originated by the activity of the voltage sensors. How-ever, this sampling frequency was not very accurately reporting the temporal courseand peak amplitude of the currents, as can be seen in the expanded trace showed inFig. 4(A). Thus, even though this sampling frequency was fast enough to record theslowly activating ion currents (Figs. 1 and 2), it was clearly inadequate to properlyresolve gating currents. Therefore, we decided to increase the sampling frequencyto 200 kHz and to apply shorter pulses (5 ms). As a consequence, we were able torecord smoother nonlinear capacitative currents and of higher amplitude (Fig. 4(B)),which allowed us to properly investigate gating currents. This is to our knowledgethe first evidence for intramembrane charge movement associated with endogenousK+ channel activity in the HEK-293 cell line.

The HEK-293 cell that we present in Fig. 4 was investigated under similar ex-perimental conditions as in Fig. 2 (TEA/Cs+, solid triangles), which means, in thepresence of internal solution F and external solution D (Table I). For a more accurateexamination of gating currents, we decided to supplement the external solution Dwith a combination of CdCl2 plus LaCl3 in order to block any possible influx of Ca2+

ions through endogenous voltage-gated Ca2+ channels (Avila and Dirksen, 2000). Itshould be mentioned however that we were unable to detect significant Ca2+ currentsduring the test pulses in the presence of 5 mM extracellular Ca2+ (Fig. 2 TEA/Cs+,solid triangles). Nevertheless, any contribution of possible tail Ca2+ currents to thecapacitative transient at the “off” of the test pulses will be expected to result in anoverestimation of the intramembrane charge movement. Thus, we decided to includethese inorganic Ca2+ channels blockers. Figure 5(A) shows representative gating cur-rents recorded at potentials from −50 to +60 mV. The average voltage-dependence

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Fig. 5. Analysis of endogenous intramembrane charge movement. (A) Nonlinear capacitativecurrents recorded with external solution D and internal solution F. Test pulses of 5 ms and variableamplitude (Vm, mV) were delivered from an HP of−80 mV and the resulting current traces digitizedevery 5 µs (200 kHz). (B) Average voltage-dependence of intramembrane charge movement. Theintegral (Q) of nonlinear capacitative currents (recorded as in A) at the “on” (Qon, open circles)and at the “off” (Qoff, solid circles) of the test pulses was calculated, normalized for its respectivemembrane capacitance (Cm), and plotted as a function of the membrane potential during the testpulse (Vm). Experimental data from each cell were fitted according to a Boltzmann equation (seeMaterials and Methods section), and the resulting kinetic parameters are given in Table II. (C)Correlation between maximal intramembrane charge movements at the “off” (Qmax-off, verticalaxis) and the “on” (Qmax-on, horizontal axis) of the test pulses. A linear regression of the experi-mental data (continuous line) resulted in a correlation factor (r) of 0.77. (D) Histogram distributionfor the Qmax-on values illustrated on (C) (n = 13). The superimposed continuous line representsa Gaussian distribution curve that was generated by using the average Qmax-on values (10.0 ± 1.0nC/µF).

for the charge movements (Q–V curves) estimated at the “on” (Qon) and the “off”(Qoff) are being plotted in Fig. 5(B). A very interesting finding is that Qoff exhibits alower threshold for activation (∼−40 mV vs.∼−30 mV), compared to Qon (Fig. 2(A)and (B)). The Q–V curves were fitted according to a Boltzmann equation and the

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Table II. Parametters of Fitted Q–V Curves

n Qmax (nC/µF) V1/2 (mV) k (mV)

Qon 13 10.0 ± 1.0 −2.3 ± 2.0∗ 15.7 ± 0.6Qoff 13 10.1 ± 1.5 −21.5 ± 1.7 13.6 ± 0.7

∗ p < 0.001.

resulting kinetic parameters are shown in Table II. As can be seen in the Q–V curvesand Table II, there was a significant shift (∼−20 mV) in the voltage for half maximalactivation (V1/2) of the charge movement at the “off” of the test pulses, compared tothe charge movement at the “on”, in the absence of significant changes in either theslope factor (k) or the maximal charge movement (Qmax). Thus, the lower thresholdfor activation of Qoff could be solely explained by a selective shift of the V1/2 valuesto more hyperpolarized potentials. As mentioned before, there are no significant dif-ferences between the average Qmax-on and Qmax-off values. Furthermore, the Qmax-on

and Qmax-off values that were recorded from each cell exhibited a high correlation,as can be seen in Fig. 5(C). Lastly, the Qmax values that were obtained from differ-ent cells were plotted as a histogram, and the resulting plot was well described bya Gaussian distribution curve (Fig. 5(D)). Taken together, the results from Figs. 4and 5 indicate that HEK-293 cells exhibit endogenous gating currents, which displaya normal distribution, and posses different V1/2 values for activation at the “on” andthe “off” of the test pulses.

DISCUSSION

The expression of recombinant proteins in heterologous systems has proved tobe a powerful tool for characterizing membrane transport systems. In particular, theadvantages and possibilities of several cell lines have been exploited to advance theknowledge on the physiology of neuronal membrane ion channels. However, preciseunderstanding of the properties of endogenous currents is a necessary preconditionfor studies of ion channel expression in these cells. The HEK-293 cell line is oneof the most widely used mammalian cell expression systems. Many voltage-gatedion channel subunits have been heterologously expressed in these cells for differ-ent purposes. Though native ion currents in untransfected HEK-293 cells have beendocumented, it has been generally assumed that the expression of endogenous chan-nels is negligible. Therefore, we sought to determine what would be the contributionof the different types of native channels to the whole-cell current in this cell line.The main aim of our project was to investigate the functional expression of differ-ent endogenous voltage-gated ion channels, and to characterize for the first timethe biophysical properties of the gating currents elicited by endogenous ion channelactivity.

It is worth mentioning that it has been recently documented that HEK-293 cellshave an unexpected relationship to neurons. Shaw et al. (2002) found that this cell lineexpresses significant levels of mRNA and protein for several major neurofilamentsubunits which are found only in neurons, suggesting that the HEK-293 cells belong

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to the neuronal lineage. Consistent with this view, by using cDNA microarray analysisthese authors also reported striking similarities between the gene expression profilein HEK-293 cells and neuronal stem cells. Notably, one of the conclusions of this workis that HEK-293 cells should not be considered typical kidney cells and should notbe used as kidney controls or to study normal kidney-related functions. Similarly, theuse of this cell line as a nonneuronal control may also be inappropriate given theirattributes of developing neurons and neuronal progenitor cells. Lastly, consistentwith a neuronal phenotype, our electrophysiological studies show that HEK-293cells express at least two types of endogenous voltage-gated K+ channels which, asdiscussed below, are key elements in neuronal signaling.

The understanding of the nervous system at the cellular level requires detailedinformation on the intrinsic biophysical properties of neurons, muscles, and thesynaptic connections they make. Electrophysiological studies of neurons have re-vealed a diverse array of voltage-gated ion channels. Among them, K+ channelsrepresent one of the most important classes in terms of function. These proteinsestablish the resting membrane potential, modulate the frequency and duration ofaction potentials, and are targets of several chemical messengers. Consequently, anunderstanding of K+ channel function at the molecular level is of great practical in-terest for neuroscientists. In the current report, gating currents arising entirely fromendogenous K+ channel activity were unambiguously resolved for the first time innative HEK-293 cells under standard culture conditions, providing valuable infor-mation concerning to the mechanisms underlying the activation of voltage-gated K+

channels.Likewise, although certain levels of voltage-gated Ca2+ and Cl− channel ex-

pression have been reported in the HEK-293 cell line (Berjukow et al., 1996; Zhuet al., 1998), a contribution from these types of channels to the whole-cell ionic currentrecorded can be practically excluded from our patch-clamp studies (Figs. 1 and 2). Thereason for this discrepancy is unclear; possibly the clone of HEK-293 cells used in ourstudies is different to those employed in the investigations of endogenous Ca2+ andCl− channels or perhaps the number of the cell culture passages may be importantlydifferent. In addition, these factors combined with possible differences in experi-mental designs might explain why other investigators have been unable to observesignificant levels of endogenous charge movements in HEK-293 cells (Bangaloreet al., 1996; Jones et al., 1997; Josephson, 1997). In support to this interpretation, it isworth mentioning that the density of the endogenous Ca2+ channels in the HEK-293cells seems to be dependent on the cell culture conditions and particularly on thepresence of fetal bovine serum (FBS) (Berjukow et al., 1996). As for our experiments,cells were kept in a FBS-free culture medium (see Materials and Methods section).However, though the cell culture conditions and duration (passage) cannot be ruledout as a potential source for some discrepancies observed in the biophysical profileof endogenously expressed outward currents in HEK-293 cells, experimental evi-dence indicates that, at least in some cases, differences in these variables (i.e. cultureconditions and duration) cannot fully explain such discrepancies. For example, Yuand Kerchner (1998) reported similar native K+ currents between HEK-293 cellsat passage 31 and HEK-293 cells directly obtained from the ATTC. Furthermore,though Jiang et al. (2002) reported similar results from HEK-293 cells at passage 31

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(compared to those from Yu and Kerchner, 1998), they also showed evidence for aK+ current of the A type, not previously described. In our studies, cells were keptin standard culture conditions and the electrophysiological recordings were madeat passages 30–35. Notably, the pharmacological and biophysical profile of the K+

outward currents, except for the absence of the A-type current described by Jianget al. (2002), were comparable to that observed in previous studies in terms of levelof expression, macroscopic kinetics, and sensitivity to tetraethylammonium (TEA)chloride. However, as mentioned above the possibility exists that in the HEK-293cell line the native K+ currents may change upon passage. This hypothesis would bean interesting topic for future studies.

Regardless of the reasons behind these differences, the results presented sug-gest important differences in the regulation of the expression of voltage-gated K+

and Ca2+ channels. As mentioned earlier, among other important cell functions K+

channels play a critical role setting the resting membrane potential. Therefore, itwould not be unreasonable to expect that in HEK-293 cells, as in many other celltypes, the expression of some types of K+ channels appear to be enabled by default.On the other hand, Ca2+ channels play more specialized roles and therefore theirexpression seems to require one or more specific signals (apparently not presentwithin the equine serum). It should be noted that morphological and neurochemicaldifferentiation in many cell lines are accompanied by changes in the expression ofCa2+ channels, and that a role for the cAMP signaling pathway has been involvedin this process (Kushmerick et al., 2001). It is also worth mentioning that the typeof Ca2+ channel being regulated depends on the chemical messenger. For example,in the neuroendocrine GH3 cell line it has been found that epidermal growth fac-tor (EGF) increases high-threshold current density, but has no significant effect onthe current density through low-threshold Ca2+ channels. In contrast, insulin andglucocorticoids selectively modify low-threshold channel expression (Meza et al.,1994).

As mentioned earlier, this is the first study of gating currents due to endogenousion channel activity in the HEK-293 cell line. The gating currents elicited during K+

channel activation exhibited interesting biophysical features. Gating charge move-ment at the “on” of the test pulses appears inhibited at modest test depolarizations,a result that is evident in the traces at −40 and −30 mV, illustrated in Fig. 5(A).At more depolarized potentials the asymmetry in the amplitude of the gating cur-rents at the “on” and the “off” of the test pulses is less, as if all the gating chargeat the “on” could only be moved with sufficiently strong depolarizations. The Q–Vcurves shown in Fig. 5(B) and (C) confirm this proposal: the maximal values for thecharge movements at the “on” (Qmax-on) and the “off” (Qmax-off) of the test pulsesare practically the same, but a∼20 mV hyperpolarizing shift in Qoff–V was observed(Fig. 5, see also Table I). These findings suggest the existence of kinetic differencesfor the voltage-dependent transitions of voltage sensors leading to the activation anddeactivation states. Hence, although Qon is smaller than Qoff at negative potentials,this discrepancy is unlikely to arise from a technical artifact, because the high corre-lation between the Qmax-on and the Qmax-of values (Fig. 5(C)). Rather, the differencein Qon and Qoff at threshold potentials argues that part of the gating charge of closedchannels moves very slowly during activation due to slow rate constants leading to

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the open state of the channels. However, all the gating charge moved during thetest depolarization should be detectable during repolarization (Qoff) because rateconstants leading to deactivation of the channels may be rapid. Thus our results areentirely consistent with the idea that the voltage sensors of endogenous K+ channelsin HEK-293 cells are capable to undergo slow transitions to the activated state atthreshold membrane potentials but fast transitions to the deactivated state at anymembrane potential.

The present results, which describe the basic biophysical features of the gatingcurrents of endogenously expressed K+ channels in HEK-293 cells, may be com-pared with work reported previously for various types of recombinant K+ channels(Bezanilla et al., 1994; Perozo et al., 1994; Stefani et al., 1997). In addition, consis-tent with the general structural homology with other type of voltage-gated channels,there are also some similarities with the gating currents recorded form HEK-293cells expressing recombinant Ca2+ channels (Bangalore et al., 1996; Jones et al.,1997; Josephson, 1997). However, a new and important finding of this study is theidentification of two major components of the gating currents in the HEK-293 cellsarising from K+ channel activity. As discussed above, these components of the chargemovement differ on their kinetic features as well as by their voltage dependence. Atthe onset of depolarization, one of the components was slow and activated at morenegative membrane potentials, and we anticipate that it comprises a small fractionof the total charge movement. In contrast, the second component was activated atmore positive potentials and comprised most of the total charge movement. Theseresults provide new insights into the gating mechanisms for native K+ channels inHEK-293 cells and support the notion that these cells express at least two differenttypes of voltage-gated K+ channels.

Our results are also in agreement with the transcriptional expression of differ-ent genes coding K+ channels from HEK-293 cDNA (Gamper et al., 2002; Jianget al., 2002). They indicate that the endogenous outward current detected in HEK-293 cells is carried through at least two different classes of K+-selective channels. Asignificant proportion of the whole-cell current was suppressed by the K+ channelblocker TEA. This current displayed a fast activation phase and inactivated slowly.These features implicate delayed rectifiers channels as mediators of this current. Inaddition, the presence of a fast activating and slow inactivating TEA-insensitive com-ponent of the whole-cell current was also detected in the HEK-293 cells. This currentwas eliminated by replacing internal K+ with Cs+ and seemed to activate at positivevoltages, resembling in many respects a Ca2+-dependent K+ current. Interestingly,Jiang et al. (2002) have recently reported that multiple voltage-gated K+ channelgenes are expressed in native HEK-293 cells, including those coding several delayedrectifier channels. However, it is difficult to establish which of the K+ channel genesgenerate the native current in HEK-293 cells, since the expression of the genes notnecessarily lead to the translation of the corresponding K+ channel proteins. In ad-dition, the functional K+ channel complex may be composed of the heterogeneousassembly of different K+ channel ion-conducting α subunits. Moreover, the endoge-nous expression of the different genes encoding the Ca2+-dependent K+ channelshas not been explored yet. Therefore, there are no ready answers with respect to themolecular identity of endogenous K+ channels in the HEK-293 cell line, though the

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use of specific antibodies and the knockdown of the currents by K+ channel subunit-specific antisense oligonucleotides will help to determine the relative expression ofthe distinct K+ channel subunits.

Lastly, electrophysiological studies involving heterologous expression of neu-ronal channels should be expected to result in ionic and gating currents easily identi-fiable and distinguishable from any endogenous current attributable to the expressionsystem. Thus, an expression system exhibiting low levels of endogenous channels willbe very useful to simplify the interpretation of these studies. For this reason, a bio-physical characterization of the endogenous channels is required. Our results showthat, in standard culture conditions, neither voltage-gated Ca2+ nor Cl− channelsare apparently present on the plasma membrane of native HEK-293 cells. Theseresults indicate that the cell line HEK-293 is a suitable expression system for futurestudies accessing the functional properties of both Ca2+ and Cl− channels. In con-trast, though the HEK-293 cell line has provided and will provide new insights onneuronal K+ channel function and regulation, precaution needs to be taken becauseof the presence of endogenous channels that may interfere with the study of theexogenously expressed channels. A better understanding of endogenous channelsand the optimization of experimental conditions (i.e. voltage protocols, the use ofspecific pharmacological agents, etc.) may help to minimize potential problems in thecharacterization of heterologously expressed neuronal K+ ion conducting proteins.It must be considered, as previous studies have shown, that the endogenously ex-pressed K+ channel subunits may regulate the heterologous expression of some othervoltage-gated K+ channel subunits and drastically change the biophysical propertiesof the expressed currents (Petersen and Nerbonne, 1999; Uebele et al., 1996). Thesefindings highlight the importance of understanding the molecular background of ex-pression systems used in K+ channel expression studies. In fact, one can anticipatethat the actions on the whole-cell K+ current of an introduced K+ subunit may bethe result, at least in part, of the interaction with endogenous subunits. Therefore,an issue that must be resolved with all heterologous expression systems includingHEK-293 cells is whether they contain endogenous, function-altering K+ channelsubunits. This could affect the interpretation of previous and future studies on K+

channel gating, pharmacology, and trafficking.

ACKNOWLEDGMENTS

This work was supported by grants from CONACyT to G.A. and R.F. and fromthe TWAS to R.F. We thank G. Cota for allowing us to use his patch-clamp system, F.Gomez-Lagunas for helpful discussions, and J. L. Martınez for technical assistance.

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