QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.098103v1 317/2/865    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, J. Articles by Zhang, S. Search for Related Content PubMed PubMed Citation Articles by Guo, J. Articles by Zhang, S.

CARDIOVASCULAR

Molecular Determinants of Cocaine Block of Human Ether-á-go-go-Related Gene Potassium Channels

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Received November 3, 2005; accepted January 4, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The use of cocaine causes cardiac arrhythmias and sudden death. Blockade of the cardiac potassium channel human ether-á-go-go-related gene (hERG) has been implicated as a mechanism for the proarrhythmic action of cocaine. hERG encodes the pore-forming subunits of the rapidly activating delayed rectifier K⁺ channel (I_Kr), which is important for cardiac repolarization. Blockade of I_Kr/hERG represents a common mechanism for drug-induced long QT syndrome. The mechanisms for many common drugs to block the hERG channel are not well understood. We investigated the molecular determinants of hERG channels in cocaine-hERG interactions using site-targeted mutations and patch-clamp method. Wild-type and mutant hERG channels were heterologously expressed in human embryonic kidney 293 cells. We found that there was no correlation between inactivation gating and cocaine block of hERG channels. We also found that consistent with Thr-623, Tyr-652, and Phe-656 being critical for drug binding to hERG channels, mutations in these residues significantly reduced cocaine-induced block, and the hydrophobicity of the residues at position 656 dictated the cocaine sensitivity of the channel. Although the S620T mutation, which removed hERG inactivation, reduced cocaine block by 21-fold, the S620C mutation, which also completely removed hERG inactivation, did not affect the blocking potency of cocaine. Thus, Ser-620 is another pore helix residue whose mutation can interfere with cocaine binding independently of its effect on inactivation.

hERG block represents a common mechanism for drug-induced LQTS, which can potentially lead to a severe cardiac arrhythmia, torsades de pointes, and sudden cardiac death (Keating and Sanguinetti, 2001). Because of the fatal nature of LQTS side effects, a number of commonly prescribed medications have been removed from the market (Keating and Sanguinetti, 2001). The mechanisms for many therapeutically and structurally diverse compounds preferentially blocking hERG channels are not well understood and are of significant interest for drug safety. Site-directed mutagenesis has identified pore helix residues Thr-623, Ser-624, and Val-625 and aromatic residues Tyr-652 and Phe-656 in the S6 transmembrane segment of hERG subunits to be important molecular determinants for high-affinity binding to hERG channels by many drugs, such as MK-499, dofetilide, cisapride, terfenadine, quinidine, and clofilium (Lees-Miller et al., 2000; Mitcheson et al., 2000; Perry et al., 2004). In addition to the structural determinant residues, the inactivation gating of hERG seems also important for high-affinity drug binding (Ficker et al., 1998, 2001; Zhang et al., 1999). For example, the structurally related EAG channel that also has the tyrosine and phenylalanine residues equivalent to Tyr-652 and Phe-656 and has the residues equivalent to Thr-623, Ser-624, and Val-625 of hERG but does not inactivate is not sensitive to hERG blocker dofetilide. Sensitivity to block by dofetilide was introduced when the EAG channel was made capable of inactivating by mutations (Ficker et al., 2001). In addition, mutations that disrupt inactivation reduce the potency of blockade by various compounds (Ficker et al., 1998; Zhang et al., 1999). Recent studies further suggested that it is the positioning of the aromatic residues in the S6 segment, not inactivation per se, that dictates the sensitivity of hERG to cisapride (Chen et al., 2002; Lin et al., 2005).

In the present study, we investigated the role of inactivation and the structural determinants of hERG in cocaine sensitivity of the channel. Whole-cell voltage-clamp technique was used to record currents from wild-type (WT) and mutant hERG channels heterologously expressed in human embryonic kidney (HEK) 293 cells. Our results demonstrate a lack of correlation between hERG inactivation and block by cocaine. We also show that amino acid residues in pore helix and S6 segment of hERG are critical for cocaine block of the channel. Hydrophobicity of the side chain of residue 656 determines cocaine affinity. We further describe that in addition to affecting hERG inactivation gating, the S620T mutation directly interferes with cocaine binding to hERG channels.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Molecular Biology. The hERG cDNA in pCDNA3 was obtained from Dr. Gail A. Robertson (University of Wisconsin, Madison, WI; Trudeau et al., 1995). We made mutations in the pore and S6 region of hERG to assess the role of inactivation and the binding domain for cocaine block of the channel. Mutations of hERG were introduced via PCR using overlap extension (Ho et al., 1989). The forward and reverse flanking primers were designed to cover two unique restriction sites (BstEII at nucleotide 2038 and SbfI at nucleotide 3093). The first round of PCR was performed using the forward flanking primer-reverse mutant primer and the reverse flanking primer-forward mutant primer, respectively. The resulting two PCR products were used as templates and amplified by flanking primers in a second round of PCR. The final PCR product was cloned into Zero Blunt Vector (Invitrogen, Carlsbad, CA) and was digested with BstEII and SbfI (New England Biolabs, Beverly, MA). Fragments with mutations were used to replace the corresponding WT fragment in the hERG expression plasmid. The S620T, S620C, S631A, G628C: S631C, F656V:S620T, and F656V:S631A mutations were made to remove hERG inactivation gating (Smith et al., 1996; Ficker et al., 1998; Zhang et al., 1999). The F627Y and S641A mutations were constructed to accelerate inactivation to assess the role of inactivation gating in cocaine block. The T623A, S624A, and Y652A mutations were constructed to assess the role of these amino acid residues in cocaine-induced block. The F656M, F656T, F656V, F656W, and F656Y mutations were made to assess the correlation between the hydrophobicity of the side chain of residues 656 and cocaine block. The S620A, S620F, S620I, S620V, and S620Y mutations were constructed, but none of them expressed functional channels. To generate double mutations of S620T:F656V and S631A:F656V, we used BglII (New England Biolabs) to obtain a fragment that contained the S620T or S631A mutant. The fragment was used to replace the equivalent fragment in the F656V mutant channel. All mutations were verified by using a high-throughput 48 capillary ABI 3730 sequencer (UCDNA Services, University of Calgary, Calgary, AB, Canada).

Mutant hERG channels were transiently expressed in HEK 293 cells (American Type Culture Collection, Manassas, VA). HEK 293 cells were seeded at 5 x 10⁵ cells/60-mm-diameter dish. The cells were transiently transfected using 10 µl of Lipofectamine with 4 µg of hERG mutant cDNA in pCDNA3 vector. After 24 to 48 h, 50 to 80% of cells expressed channels. To evaluate the role of KCNE1 (minK) in cocaine block of hERG, HEK 293 cells were cotransfected with 4 µg of hERG mutant cDNA plus 4 µg of KCNE1 cDNA in pCDNA3 vector mixed in 10 µl of Lipofectamine. The KCNE1 cDNA in pSP64 vector was obtained from Dr. Michael C. Sanguinetti (University of Utah, Salt Lake City, UT; Sanguinetti et al., 1996). Nontransfected HEK 293 cells contain a small-amplitude background current that is usually less than 100 pA upon a depolarizing pulse to 50 mV. Thus, the effects of overlapping endogenous currents of HEK 293 cells on the expressed current are minimal (Lin et al., 2005). An HEK 293 cell line stably expressing hERG channels obtained from Dr. Craig T. January (University of Wisconsin; Zhou et al., 1998) was also used. In this cell line, the hERG cDNA (Trudeau et al., 1995) was subcloned into BamHI/EcoRI sites of the pCDNA3 vector (Invitrogen). The stably transfected cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum and contained 400 µg/ml Geneticin (G418) to select for transfected cells. For electrophysiological study, the cells were harvested from the culture dish by trypsinization and stored in standard minimal essential medium at room temperature for later use. Cells were studied within 8 h of harvest.

Patch-Clamp Recording Method. The whole-cell voltage-clamp method was used. The pipette solution contained 135 mM KCl, 5 mM EGTA, 1 mM MgCl₂, and 10 mM HEPES and was adjusted to pH 7.2 with KOH. The bath solution contained 135 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, 1 mM MgCl₂, and 2 mM CaCl₂ and was adjusted to pH 7.4 with NaOH. For recordings in the presence of different external K⁺ concentrations, the concentration of Na⁺ was changed according to K⁺ concentration to maintain a constant osmolarity, and the pH was adjusted to 7.4 with NaOH or KOH. E-4031 was purchased from Calbiochem (EMD Biosciences, San Diego, CA). Cocaine was purchased from Sigma-Aldrich (St. Louis, MO). Cisapride and all other chemicals were from Sigma-Aldrich (Oakville, ON, Canada). Cocaine was either dissolved in the water to make 10 to 100 mM stock solutions or was added to the bath solutions to make desired concentrations.

Aliquots of cells were allowed to settle on the bottom of a <0.5-ml cell bath mounted on an inverted microscope (TE2000; Nikon, Tokyo, Japan). Cells were superfused with specific bath solutions. The bath solution was constantly flowing through the chamber, and the solution was changed by switching the perfusates at the inlet of the chamber. This bath solution change took less than 10 s. Patch electrodes were fabricated using thin-walled borosilicate glass (WPI, Sarasota, FL). The pipette had inner diameters of 1.5 µm and resistances of 2 M when filled with the pipette solution. An Axopatch 200B amplifier was used to record membrane currents. Computer software (pCLAMP9; Axon Instruments, Foster City, CA) was used to generate voltage-clamp protocols, acquire data, and analyze current signals. Data were filtered at 5 to 10 kHz and sampled at 20 to 50 kHz for all protocols. Typically, 80% series resistance (R_s) compensation was used, and leak subtraction was not used. Concentration effects were quantified by fitting the data to the Hill equation (I_drug/I_control = 1/[1 + (D/IC₅₀)^H], where D is the drug concentration, IC₅₀ is the drug concentration for 50% block, and H is the Hill coefficient to the results. Data are given as mean ± S.E.M. Clampfit (Axon Instruments) and Origin (OriginLab Corp., Northampton, MA) were used for data analysis. Curve fitting was done using multiple nonlinear least-squares regression analysis. All experiments were performed at room temperature (23 ± 1°C).

	Results

Top Abstract Materials and Methods Results Discussion References

 
External K⁺ Concentration Does Not Affect Cocaine Block of hERG Channels. Changes in extracellular K⁺ concentration () have been shown to influence hERG inactivation gating and the sensitivity of I_Kr/hERG to drugs such as dofetilide, E-4031, and cisapride (Yang and Roden, 1996; Wang et al., 1997; Lin et al., 2005). To investigate the effects of on cocaine block of hERG, cocaine effects were examined in 0, 5, or 135 mM conditions. To record hERG currents, the hERG-expressing HEK cells were repetitively depolarized to voltages between -70 and 70 mV for 4 s, followed by a step to -50 mV to record tail currents. The holding potential was -80 mV. We found that the cocaine block of hERG was not -dependent. Bath application of 10 µM cocaine blocked 50% of the currents in 0, 5, or 135 mM (data not shown). To obtain concentration-dependent relationships for block of hERG by cocaine, six to eight cells were studied at each drug concentration. The blocking effects were measured by the degree of peak tail current suppressions at each cocaine concentration. The normalized data were plotted against cocaine concentration and fitted to the Hill equation. The half-maximal inhibition concentration (IC₅₀) for cocaine block of hERG current was 11.1 ± 1.4 µM at 0 , 8.7 ± 1.6 µM at 5 mM , and 14.4 ± 1.9 µM at 135 mM . The IC₅₀ values are not significantly different from one another (n = 6-8 cells for each data point; p > 0.05). The corresponding Hill coefficient was 1.2, 1.0, and 0.9, respectively, suggesting that occupation of a single binding site accounts for the block by cocaine.

It has been reported that KCNE1 (minK) may contribute to the formation of native I_Kr channels (McDonald et al., 1997). To study whether KCNE1 plays a role in cocaine-induced hERG block, we coexpressed hERG and minK in HEK 293 cells and analyzed the effects of cocaine. Successful expression of KCNE1 in these cells were approved in separate experiments by expressing KCNQ1 (KvLQT1) alone or KCNQ1 plus KCNE1. Expression of KCNQ1 alone generated a fast activating K⁺ current. In contrast, coexpression of KCNQ1 + KCNE1 consistently gave rise to a larger, slowly activating current, which represents I_Ks (Zhang et al., 2001; Zhang, 2005). We found that coexpression of KCNE1 with hERG did not affect cocaine block of the channel. The IC₅₀ and Hill coefficient for cocaine block of current because of hERG + KCNE1 coexpression were 6.7 ± 1.0 µM and 1.0 (at 5 mM ; n = 3). These values are not different from cocaine block of currents due to hERG expression.

Effects of Inactivation-Modified Mutations on Cocaine Block of hERG Channels. hERG inactivation has been shown to play an important role in high-affinity drug binding to hERG channels (Ficker et al., 1998, 2001; Zhang et al., 1999). We assessed the role of inactivation in hERG block by cocaine. Three well documented inactivation-deficient mutant hERG channels, S620T, S631A, and G628C:S631C, were constructed. Previous studies have shown that these mutations interfere with hERG C-type inactivation and significantly reduce blocking potency of various drugs (Ficker et al., 1998; Zhang et al., 1999). As shown in Fig. 1, these mutations essentially removed hERG inactivation. Figure 1A shows families of the mutant currents for control conditions and after exposure to 10 µM cocaine. From a holding potential of -80 mV, depolarizing steps were applied for 4 s to voltages between -70 and +70 mV in 10-mV increments. The depolarizing steps were followed by a repolarization step to -50 mV to record tail currents. In these mutant channels, depolarizing steps induced outward currents that increased in amplitude as depolarizing steps became more positive. The inward rectification seen in WT hERG channels was absent. Application of 10 µM cocaine did not affect the S620T current. However, it blocked the S631A and G628C:S631C currents by approximately one-half. To quantify cocaine block, the current amplitude at the end of a 4-s depolarizing step to 50 mV under each cocaine concentration was normalized to the control value. Four to nine cells were studied at each concentration, and the normalized current amplitudes were plotted against cocaine concentration. The data were fitted to the Hill equation. The IC₅₀ for cocaine block of the S620T, S631A, or G628C:S631A current was 178.6 ± 55.0 µM (n = 9), 12.4 ± 3.2 µM (n = 7), or 9.2 ± 1.5 µM (n = 4), respectively. The corresponding Hill coefficient was 1.0, 1.0, and 0.9. Thus, among three inactivation-deficient mutants, only the S620T mutation reduced cocaine block (n = 9; p < 0.01 compared with WT channels). The S631A and G628C:S631C mutations did not affect channel sensitivity to cocaine (p > 0.05 compared with WT channels).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Effects of inactivation-deficient mutations on the cocaine block of hERG channels. A, S620T, S631A, and G628C:S631C hERG currents in control conditions and after exposure to 10 µM cocaine. The intracellular solution contained 135 mM K+ and the bath solution contained 5 mM K+. B, concentration dependence of cocaine block of S620T, S631A, and G628C:S631C channels. Each data point represents the average of four to seven cells. Cocaine block of WT channels is shown for comparison.

To further investigate the role of the inactivation in cocaine block, we constructed the F627Y and S641A mutations. The S641A mutation has been reported to accelerate the onset of hERG inactivation (Bian et al., 2004). We discovered that the F627Y mutation also significantly accelerates hERG inactivation. Because of the accelerated inactivation, the outward current in 5 mM was very small. Therefore, the F627Y and S641A mutant currents were recorded in 135 mM . To examine the inactivation time course of these currents, mutant hERG channels were fully activated and inactivated by a depolarizing step to +60 mV for 500 ms. The cell was then repolarized to -100 mV for 10 ms to allow channel recovery from inactivation to open state but not enough for channel deactivation (Smith et al., 1996; Spector et al., 1996; Zhang et al., 2003). A test step was then applied to different voltages to observe inactivation time courses (Fig. 2A). The inactivation time constant (_inact) was obtained by fitting the current decay to a single exponential function. Figure 2A shows WT hERG currents during the test steps to voltages between 10 and 60 mV in 10-mV increments in 135 mM . Figure 2B shows the F627Y hERG currents under the same conditions. The averaged _inact values for WT, F627Y, and S641A currents in five to eight cells are plotted as a function of test potentials in Fig. 2C. Both the F627Y and S641A mutations significantly accelerated the onset of inactivation at all voltages tested. For example, at 20 mV, the _inact for WT was 17.9 ± 1.2 ms (n = 6, in 135 mM ), whereas it was 2.3 ± 0.2 ms for F627Y channels (n = 6,, in 135 mM ; p < 0.01), and it was 2.4 ± 0.1 ms for S641A channels (n = 4; p < 0.01). In contrast to the accelerated inactivation, the activation properties of both F627Y and S641A mutant channels were only slightly different from WT channels. The voltages for half-maximal activation (V_1/2) and the slope factor (k) of WT channels were -10.7 ± 1.1 and 8.4 ± 1.0 mV (n = 6). The V_1/2 and k of the F627Y channels were -18.5 ± 1.1 mV (n = 6; p < 0.05) and 10.2 ± 0.4 mV (p > 0.05). The V_1/2 and k of the S641A channels were -9.7 ± 2.0 and 7.9 ± 0.3 mV (n = 4; p > 0.05). Figure 2, D and E, shows families of F627Y currents in control (D) and in the presence of 10 µM cocaine (E). Figure 2F compares the concentration-dependent block of WT, F627Y, and S641A currents by cocaine. The IC₅₀ and Hill coefficient of cocaine block of F627Y were 7.7 ± 1.4 µM and 0.9 and for the S641A were 12.9 ± 0.6 µM and 0.8 (n = 4-7 cells for each data point). Thus, IC₅₀ of cocaine block of F627Y or S641A was not different from that on WT channels (IC₅₀ = 14.4 ± 1.9 µM; Hill coefficient = 0.9, in 135 mM ; p > 0.05). The fact that F627Y and S641A accelerated hERG inactivation but did not affect the potency of cocaine block suggests that inactivation plays no role in cocaine-hERG channel interactions.

View larger version (26K): [in this window] [in a new window]   Fig. 2. F627Y and S641A accelerated inactivation but did not affect the cocaine block of hERG channels. Whole-cell hERG currents were recorded in symmetric K+ concentrations (135 mM). A, voltage protocol and WT hERG inactivation time courses. B, F627Y inactivation time courses. Dotted lines in A and B indicate the zero current level. C, inact-voltage relationships in WT (), F627Y (), and S641A hERG channels (). D and E, voltage protocol and families of F627Y currents recorded in the absence (D) and presence of 10 µM cocaine (E). F, concentration-dependent block of WT (), F627Y (), and S641A hERG channels (). The block was calculated based on the degree of the tail current suppression at -50 mV relative to the control value (n = 4-7; p > 0.05).

View larger version (37K): [in this window] [in a new window]   Fig. 3. Competitive binding of cocaine with cisapride and E-4031. A, amplitudes of hERG tail currents during cisapride (1 µM) block and upon washout. B, amplitudes of hERG tail currents during cisapride block followed by cocaine (100 µM) application and upon washout. C, recovery of the hERG current in 2 min of washout after 1 µM cisapride or after 1 µM cisapride followed by 100 µM cocaine. D, amplitudes of hERG tail currents during E-4031 (0.5 µM) block and upon washout. E, amplitudes of hERG tail currents during E-4031 block followed by cocaine (100 µM) application and upon washout. F, recovery of the hERG current in 2 min of washout after 0.5 µM E-4031 or after 0.5 µM E-4031 followed by 100 µM cocaine. Although the current recovery after 1 µM cisapride or 0.5 µM E-4031 blockade was slow and incomplete, additional exposure to cocaine (100 µM) resulted in a significantly faster recovery of hERG currents upon drug washout (**, p < 0.01 compared with the current recovery upon washout from cisapride or E-4031).

Pore Helix Residues and Aromatic Residues of S6 Are Involved in Cocaine Binding to hERG Channels. Residues in pore helix and S6 of each hERG subunit are involved in high-affinity binding of various compounds to hERG channels (Lees-Miller et al., 2000; Mitcheson et al., 2000; Perry et al., 2004). To determine whether these residues are also important for cocaine block of hERG, T623A, S624A, and Y652A mutations were constructed, and the effects of cocaine were examined (Fig. 4). Although S624A and Y652A displayed a gating behavior similar to WT channels, T623A mutation significantly accelerated inactivation. Since we have shown that has no effect on cocaine block of hERG channels, the effects of cocaine on T623A were examined in 135 mM , whereas the effects of cocaine on S624A and Y652A were assessed in 5 mM (Fig. 4A). We found that these mutations significantly reduced the channel affinity to cocaine (Fig. 4B). The IC₅₀ for cocaine block of T623A, S624A, and Y652A was 166.8 ± 19, 45.5 ± 4.4, and 309.6 ± 49 µM, respectively. The corresponding Hill coefficient was 1.0, 1.0, and 1.1, respectively (n = 4-6 cells for each data point). These IC₅₀ values are all significantly larger than those in WT channels (8.7 ± 1.6 µM in 5 mM and 14.4 ± 1.9 µM in 135 mM ; p < 0.01). Again, the reduced cocaine sensitivity was not associated with hERG inactivation gating since inactivation was accelerated in T623A, slowed in S624A, and remained the same in Y652A. Specifically, at 20 mV, the _inact was 1.4 ± 0.2 ms for T623A (n = 7; p < 0.01 compared with 17.9 ± 1.2 ms for WT channels in 135 mM ). The _inact was 18.2 ± 3.5 for S624A (n = 4; p < 0.01) and 9.4 ± 0.9 ms for Y652A (n = 4; p > 0.05 compared with 12.9 ± 1.1 ms for WT channels in 5 mM ; n = 6). The reduced cocaine affinity of S623A, S624A, and Y652A was also not associated with the altered channel activation gating. For example, the V_1/2 and k were 3.4 ± 0.5 and 10.0 ± 0.4 mV for T623A (n = 7), -8.6 ± 2.9 and 8.0 ± 0.5 mV for S624A (n = 4), and 4.1 ± 1.8 and 9.3 ± 0.6 mV for Y652A (n = 4). Thus, V_1/2 of T623A and Y652A was more positive (p < 0.01), and V_1/2 of S624A was similar to that of WT channels, which have a V_1/2 of -10.7 ± 1.1 mV and a k of 8.4 ± 1.0 mV (n = 6).

View larger version (29K): [in this window] [in a new window]   Fig. 4. T623A, S624A, and Y652A mutation reduced cocaine sensitivity of hERG channels. A, families of T623A (135 mM and 135 mM ) and S624A and Y652A hERG K+ currents (135 mM and 5 mM ) for control and in the presence of 50 or 200 µM cocaine. B, concentration-dependent block of the hERG mutant currents by cocaine. The data for WT channel (135 mM and 5 mM ) are shown for comparison.

Phe-656 has been shown to be critical for drug binding to hERG channels (Mitcheson et al., 2000). It is shown that the hydrophobicity of the side chain at this position dictates the potency for block of hERG channels by MK-499, cisapride, and terfenadine (Fernandez et al., 2004). To test whether Phe-656 is also involved in cocaine binding, we constructed five mutant channels: F656M, F656T, F656V, F656W, and F656Y. The biophysical and pharmacological consequences of these mutations were determined. All of these mutations retained relatively normal biophysical properties. Figure 5A, a and b, shows families of the mutant currents in the absence and presence of 10, 50, or 200 µM cocaine. Currents were elicited by 4-s pulses to various voltages between -70 and 70 mV from a holding potential of -80 mV. The depolarizing steps were followed by a 5-s repolarizing pulse to record tail currents. To construct the current-voltage (I-V) relationships, currents at the end of 4-s depolarizing pulses were measured and normalized to the peak value in control recordings (Fig. 5Ac). These I-V relationships were bell-shaped and peaked at voltages between -30 and 20 mV. It is known that the rectification (bell shape) of the I-V relationship of WT hERG channels results from rapid inactivation. These results indicate that these mutant channels retain a relatively normal voltage dependence of inactivation. The voltage dependence of activation of these channels was examined by determining the potentials for V_1/2 and k of each mutant channel. The tail current amplitudes at -50 mV were plotted against the depolarizing voltages and fitted to the Boltzmann function (Table 1). The V_1/2 and k of WT channels were -10.7 ± 1.1 and 8.4 ± 1.0 mV (n = 6). Although the F656W mutation caused a significant shift of V_1/2 to the negative voltages, other Phe-656 mutations only had minor effects on V_1/2, and none of the mutations significantly changed the slope factor k (Table 1). The time constants for the onset of channel activation were analyzed at -10 mV, and the time constants for deactivation were analyzed at -50 mV (Table 1). Although the deactivation was significantly accelerated by some mutations, such as F656V, there was no significant change in the onset of activation in most mutations except for F656W. The accelerated activation of F656W was probably due to the negatively shifted activation curve. The K⁺ selectivity was analyzed by measuring reversal potential of the current in each Phe-656 mutation. The reversal potential was not altered by any of these mutations (Table 1).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Effects of Phe-656 mutations on cocaine block of hERG channels. A, a and b, families of the mutant hERG K+ currents (135 mM and 5 mM ) for control and in the presence of 10, 50, or 200 µM cocaine. c, I-V relationships for control and in the presence of 10, 50, or 200 µM cocaine for each mutant channel. B, concentration-dependent block of mutant hERG currents by cocaine. The data for WT channels are shown for comparison. C, IC50 of cocaine for each of the WT and mutant hERG channels (**, p < 0.01 compared with that of WT channels).

View this table: [in this window] [in a new window]   TABLE 1 Activation properties of mutant hERG channels Currents were measured in 135 mM /5 mM .

Consistent with the Phe-656 being a key residue for cocaine binding, the cocaine sensitivity was altered in Phe-656 mutant channels. As can be seen in Fig. 5Ab, whereas 10 µM cocaine reduced the F656W and F656Y currents by one-half, 50 or 200 µM cocaine was required to achieve a similar effect on the F656M, F656V, or F656T currents. The summarized I-V relationships of each mutant channel for control (D) and in the presence of cocaine () are shown in Fig. 5Ac. Concentration-dependent relationships for cocaine block of these channels were determined by the tail current inhibition, and data are summarized in Fig. 5B. The IC₅₀ and Hill coefficient were 8.4 ± 0.8 µM and 1.0 for F656W (n = 4), 12.2 ± 1.6 µM and 1.1 for F656Y (n = 5), 31.4 ± 3.6 µM and 0.8 for F656M (n = 7), 88.9 ± 11.8 µM and 0.9 for F656V (n = 5), and 161.8 ± 24.2 µM and 0.8 for F656T (n = 7). To compare the effect of each mutation on cocaine sensitivity of the channel, the IC₅₀ for each mutation was compared with that of WT in Fig. 5C. Although F656W and F656Y did not affect and F656M only slightly decreased cocaine sensitivity, F656V and F656T significantly decreased cocaine sensitivity of the channel. It has been shown that the magnitude of two-dimensional van der Waals hydrophobic surface area of these mutant side chains follows the order of W F Y M > V > T. A close correlation between the van der Waals hydrophobic surface area for the side chain of residue 656 and channel block by cisapride, terfenadine, and MK-499 has been described previously (Fernandez et al., 2004). Our results indicate that the hydrophobicity of the side chain of residue 656 also determines the channel sensitivity to cocaine. Thus, the Phe-656 is a critical site for cocaine binding to hERG channels.

Ser-620 Is Involved in Cocaine Binding to hERG Channels. We have shown that among the inactivation-deficient mutant channels S631A, G628C:S631C, and S620T, only the S620T significantly decreased cocaine sensitivity (Fig. 1). Ser-620 is located on the pore helix and contributes to the lining of the inner pore region (Ficker et al., 1998; Mitcheson et al., 2000). In addition to Tyr-652 and Phe-656 in S6, residues in pore helix have also been shown to contribute to the formation of the drug binding domain (Mitcheson et al., 2000), and we have shown that T623A mutation, which faces the central cavity of the channels and is located next to Ser-620 by one turn of helix, disrupts cocaine binding to hERG channels. Thus, we hypothesized that S620T mutation, in addition to its well known effect of removal of hERG inactivation, may directly disrupt cocaine binding to the channel. To examine this possibility, the inactivation-deficient mutations were introduced to hERG F656V channels. Both hERG F656V:S620T and F656V:S631A channels displayed inactivation deficiency. Depolarizations to 50 mV for 4 s induced a sustained outward current with no sign of inactivation (data not shown). The cocaine block was measured at the end of 4-s depolarization and normalized to the control values. For each drug concentration, five to 11 cells were studied. Whereas removal of C-type inactivation from F656V by S631A had little effect on the channel sensitivity to cocaine, introduction of S620T mutation to F656V significantly reduced cocaine sensitivity. The IC₅₀ for cocaine block of F656V:S631A was 56.1 ± 7 µM with a Hill coefficient of 0.7 (n = 4; p > 0.05 compared with 88.9 ± 11.8 µM for F656V). The IC₅₀ for cocaine block of F656V:S620T was 1547.1 ± 266.2 µM with a Hill coefficient of 1.0 (n = 4; p < 0.01 compared with 88.9 ± 11.8 µM for F656V). These results suggest that inactivation gating plays little role in cocaine binding to HERG channels. The S620T induced reduction of cocaine block may be through interfering with cocaine binding.

To assess mutations of Ser-620 in interfering cocaine binding to hERG channels, additional mutations were constructed. S620A was made to decrease the size of the side chain. S620V and S620I were made because either valine or isoleucine is present in most other voltage-gated K⁺ channels at the equivalent position. S620F and S620Y were made because aromatic residues of phenylalanine and tyrosine on S6 are important for high-affinity drug binding. Unfortunately, none of these mutants expressed functional channels. However, the S620C did express robust currents. Importantly, the S620C completely removed hERG inactivation but retained cocaine sensitivity similar to WT channels. As can be seen in Fig. 6A, S620C currents showed no sign of inactivation. In contrast to the S620T mutation that considerably decreased cocaine block (Fig. 1A), the S620C channel did not affect the channel sensitivity to cocaine, very similar to WT channels (Fig. 6, B and C). The IC₅₀ for cocaine block of the S620C channel was 7.7 ± 1.7 µM (n = 5; Hill coefficient of 0.7 ± 0.1), which is very close to the IC₅₀ of 8.7 ± 1.6 µM for cocaine block of WT channels (p > 0.05). Since S620C completely removed inactivation without affecting cocaine block, it indicates that cocaine block of hERG is not dependent on inactivation, and the S620T may directly interfere with cocaine binding independent of inactivation gating.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Dissociation between inactivation gating and cocaine block by Ser-620 mutations. A and B, families of the S620C mutant channel in the absence (A) and presence of 10 µM cocaine (B). C, concentration-dependent block of S620C by cocaine () is shown in comparison with those of S620T () and WT channels ().

View larger version (17K): [in this window] [in a new window]   Fig. 7. Effects of inactivation-deficient mutations on E-4031 block of hERG channels. Concentration dependence of E-4031 block of S631A, G628C:S631C, S620C, and S620T are shown along with data of WT channels for comparison.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study demonstrated that inactivation gating and cocaine block of hERG channels are not coupled. First, changing extracellular K⁺ concentration, which is known to regulate hERG inactivation (Zhang et al., 2003), did not affect cocaine affinity. Second, accelerating hERG inactivation by the F627Y and S641A mutations did not change the IC₅₀ for cocaine block. Third, disrupting hERG inactivation by mutations S631A, G628C:S631A, and S620C did not affect cocaine block of the channel. Inactivation gating has been suggested to play an obligatory role in high-affinity drug binding to hERG channels (Ficker et al., 1998, 2001). Removal of hERG inactivation has been shown to reduce hERG sensitivity to many drugs, such as dofetilide, verapamil, E-4031, haloperidol, and clofilium (Suessbrich et al., 1997; Wang et al., 1997; Ficker et al., 1998; Zhang et al., 1999). Our recent data and that presented by Chen et al. have suggested that inactivation per se is not a determinant for cisapride block of hERG channels (Chen et al., 2002; Lin et al., 2005). Instead, inactivation induced a conformation that favors drug binding, and Phe-656 is involved in the inactivation-facilitated drug block (Lin et al., 2005). In the present study, we showed that removal of hERG inactivation by each of the mutations S631A, G628C:S631C, and S620C increased IC₅₀ of E-4031 by 4- to 6-fold. In contrast, the channel inactivation did not affect cocaine block of hERG channels. The reason for this discrepancy is not known but may be related to the chemical structure of cocaine.

Based on a pharmacophore model derived from a comparative molecular field analysis (Cavalli et al., 2002), a high-affinity hERG-blocking compound is generally a flexible molecule consisting of three aromatic moieties connected through a nitrogen function that is a tertiary amine throughout the whole set of molecules. The tertiary amine renders the compounds protonated at physiological pH, which may be important in conferring biological activity. In addition, polar groups attached to the phenyl ring at one end of the drug molecule can favor the activity. Cocaine is a methyl-benzoylecgonine. Its nitrogenated base can be considered as a tertiary amine (pK_a 8.6; Crumb and Clarkson, 1990), which is incorporated into a rigid ring system. Although polar groups do exist, there is only one aromatic ring in the cocaine structure. Therefore, cocaine does not possess chemical features for high-affinity hERG inhibition. Indeed, although many hERG blockers inhibit hERG current with IC₅₀ in nanomolar range, cocaine blocks hERG with an IC₅₀ of 8.7 µM.

Despite of the uncoupling between cocaine block and hERG inactivation, we found that cocaine competes with cisapride and E-4031 for binding. Consistently, the residues important for hERG blockers such as methanesulfonanilides were also involved in cocaine binding. It has been shown that the drug binding domain of hERG is comprised of amino acids located on the S6 transmembrane segment (Tyr-652 and Phe-656) and pore helix (Thr-623 and Ser-624) of the channel subunit that face the cavity of the channel (Mitcheson et al., 2000; Perry et al., 2004). We found that mutations at these positions reduced cocaine block of the channel. For Phe-656, we found that F656W, F656Y did not change and the F656M only slightly reduced cocaine sensitivity. The F656V mutation reduced cocaine block by 10-fold, and the F656T mutation reduced hERG block by 19-fold. The different cocaine affinity among these mutations at 656 could be due to the fact that the van der Waals hydrophobic surface areas of tryptophan, tyrosine, and methionine closely resemble that of phenylalanine, whereas valine is less hydrophobic and threonine is a hydrophilic molecule. Thus, our results suggest that measures of hydrophobicity at 656 dictate cocaine sensitivity, similar to that described for other drugs (Fernandez et al., 2004).

Based on our data and a three-dimensional quantitative structure-activity relationship model (Cavalli et al., 2002), we propose that the protonated nitrogen and polar groups of cocaine are oriented toward the selectivity filter end of the central cavity, and the aromatic ring of the drugs is pointed toward the intracellular opening of the inner helices. The protonated nitrogen of cocaine may interact with Tyr-652 by cation- interactions, the polar groups of cocaine may interact with the Thr-623 and Ser-624 via polar interactions, and the aromatic ring of cocaine may interact with Phe-656 by hydrophobic interactions. However, because cocaine is rigid and does not possess typical high-affinity structure for hERG block, its interaction with Phe-656 may be weak. For example, although the F656T mutation reduced the potency of MK-499 and cisapride for hERG block by 209- and 145-fold, the same mutation F656T only reduced cocaine block by 19-fold. We have previously shown that the inactivation-associated conformational change of Phe-656 is involved in the inactivation-facilitated cisapride block (Lin et al., 2005). Therefore, it seems possible that the binding stereometry of rigid cocaine molecule to hERG channel does not allow inactivation associated conformational change to increase cocaine affinity.

The relatively weak interaction between cocaine and hERG also may explain the very rapid blocking and unblocking kinetics (Zhang et al., 2001). For many drugs, the development of hERG channel block is slow (Snyders and Chaudhary, 1996; Zhang et al., 1999). Recovery from block by drugs such as methanesulfonanilides (e.g., E-4031, dofetilide, and MK-499) is extremely slow and is thought to be due to trapping of the charged drug moiety within the inner vestibule of the channel (Mitcheson et al., 2000). If unblocking of cocaine is faster than channel closing, such kinetics would minimize trapping of cocaine.

Although we showed that cocaine block of hERG is independent of hERG inactivation, our results showed that the S620T mutation, which removes inactivation, reduced cocaine block by 21-fold. We also found that the S620T produced a much stronger effect on reduction of E-4031 block than other inactivation-deficient mutant channels. Notably, although both S620C and S620T did not inactivate, S620T reduced E-4031 block 13-fold more than S620C. The effect of S620C mutation on reduction of E-4031 potency was similar to those of S631A and G628C:S631C inactivation-deficient mutants. These results indicate that in addition to via inactivation, S620T also reduces drug block by direct interference with drug binding. This deliberation is consistent with previous studies in EAG channels. EAG channels are not sensitive to hERG blockers. In bEAG, the residues equivalent to hERG Ser-620 and Ser-631 are Thr-432 and Ala-443. Thus, with respect to the serine residues, the WT bEAG is equivalent to the hERG S620T:S631A double mutation. Ficker et al. (2001) have shown that introducing Ser at 432 (T432S) in bEAG (equivalent to position 620 in hERG) increased dofetilide sensitivity by 4-fold. Although the 4-fold increase of dofetilide block induced by the T432S was not dramatic, the fact that this mutation increased drug sensitivity but did not change the channel gating suggests that Ser at 432 (Ser-620 in hERG) was involved in the dofetilide binding to EAG channels independent of inactivation gating (Ficker et al., 2001).

Since Ser-620 is located in the pore helix, just one turn of helix next to Thr-623, it is possible that S620T disrupted drug binding by changing the conformation of the binding domain. The fact that S620T reduced cocaine sensitivity, whereas S620C did not, suggests the size of the side chain at this position is critical for the proper shape/size for drug-hERG interaction.

The concentrations for cocaine to block hERG channels are within the range achieved in humans. For example, in cocaine-associated sudden death, average post-mortem plasma cocaine concentrations of 20 µM have been reported (Mittleman and Wetli, 1984). It has been assumed that KCNE2 (MiRP1) and KCNE1 (minK) contribute to the formation of native I_Kr channels. However, we found that coexpression of KCNE1 with hERG did not affect cocaine block of the channel. As for KCNE2, it has been reported that the pharmacological sensitivity of hERG currents in Chinese hamster ovary cells was indistinguishable from that of I_Kr and was unaffected by KCNE2 (MiRP1) coexpression (Kamiya et al., 2001; Weerapura et al., 2002). Since the values of IC₅₀ for cocaine to block I_Kr and hERG are very close (Clarkson et al., 1996; O'Leary, 2001; Zhang et al., 2001), it seems that both KCNE1 and KCNE2 do not play a significant role in cocaine-induced block of hERG currents.

In summary, we have identified critical amino acid residues of hERG that contribute to cocaine binding and demonstrated dissociation between inactivation gating and cocaine block of hERG channels. The finding that mutations in Ser-620 residue affect drug binding prompts caution using the S620T mutation to evaluate the role of inactivation in drug block of hERG channels.

	Acknowledgements

 
We thank Dr. Craig T. January (University of Wisconsin) for the stable hERG cell line, Dr. Gail A. Robertson (University of Wisconsin) for the hERG cDNA, and Dr. Michael C. Sanguinetti (University of Utah) for the KCNE1 cDNA. Technical assistance by Wentao Li is also acknowledged.

	Footnotes

 
This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Manitoba and from Manitoba Health Research Council to S.Z., a recipient of the New Investigator Award from the Heart and Stroke Foundation of Canada.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.098103.

ABBREVIATIONS: LQTS, long QT syndrome; I_Kr, cardiac rapidly activating delayed rectifier K⁺ current; hERG, human ether-á-go-go-related gene; MK-499, (+)-N-[1'-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4'-piperidin)-6-yl]methanesulfonamide] monohydrochloride; EAG, ether-á-go-go; bEAG, bovine ether-á-go-go; WT, wild-type; HEK, human embryonic kidney; PCR, polymerase chain reaction; E-4031, 1-[2-(6-methyl-2-pyridyl)ethyl]-4-(methylsulfonyl-aminobenzoyl) piperidine; I-V, current-voltage.

Address correspondence to: Dr. Shetuan Zhang, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6. E-mail: szhang{at}sbrc.ca

	References

Top Abstract Materials and Methods Results Discussion References

 

Bian JS, Cui J, Melman Y, and McDonald TV (2004) S641 contributes to HERG K⁺ channel inactivation. Cell Biochem Biophys 41: 25-40.[CrossRef][Medline]

Cavalli A, Poluzzi E, De Ponti F, and Recanatini M (2002) Toward a pharmacophore for drugs inducing the long QT syndrome: insights from a CoMFA study of HERG K⁺ channel blockers. J Med Chem 45: 3844-3853.[CrossRef][Medline]

Chen J, Seebohm G, and Sanguinetti MC (2002) Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of HERG and Eag potassium channels. Proc Natl Acad Sci USA 99: 12461-12466.[Abstract/Free Full Text]

Clarkson CW, Xu YQ, Chang C, and Follmer CH (1996) Analysis of the ionic basis for cocaine's biphasic effect on action potential duration in guinea-pig ventricular myocytes. J Mol Cell Cardiol 28: 667-678.[CrossRef][Medline]

Crumb WJ Jr and Clarkson CW (1990) Characterization of cocaine-induced block of cardiac sodium channels. Biophys J 57: 589-599.[Medline]

Fernandez D, Ghanta A, Kauffman GW, and Sanguinetti MC (2004) Physicochemical features of the HERG channel drug binding site. J Biol Chem 279: 10120-10127.[Abstract/Free Full Text]

Ficker E, Jarolimek W, and Brown AM (2001) Molecular determinants of inactivation and dofetilide block in ether-á-go-go (EAG) channels and EAG-related K⁺ channels. Mol Pharmacol 60: 1343-1348.[Abstract/Free Full Text]

Ficker E, Jarolimek W, Kiehn J, Baumann A, and Brown AM (1998) Molecular determinants of dofetilide block of HERG K⁺ channels. Circ Res 82: 386-395.[Abstract/Free Full Text]

Gamouras GA, Monir G, Plunkitt K, Gursoy S, and Dreifus LS (2000) Cocaine abuse: repolarization abnormalities and ventricular arrhythmias. Am J Med Sci 320: 9-12.[CrossRef][Medline]

Ho SN, Hunt HD, Horton RM, Pullen JK, and Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51-59.[CrossRef][Medline]

Kamiya K, Mitcheson JS, Yasui K, Kodama I, and Sanguinetti MC (2001) Open channel block of HERG K⁺ channels by vesnarinone. Mol Pharmacol 60: 244-253.[Abstract/Free Full Text]

Keating MT and Sanguinetti MC (2001) Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104: 569-580.[CrossRef][Medline]

Kloner RA, Hale S, Alker K, and Rezkalla S (1992) The effects of acute and chronic cocaine use on the heart. Circulation 85: 407-419.[Abstract/Free Full Text]

Lees-Miller JP, Duan Y, Teng GQ, and Duff HJ (2000) Molecular determinant of high-affinity dofetilide binding to HERG1 expressed in Xenopus oocytes: involvement of S6 sites. Mol Pharmacol 57: 367-374.[Abstract/Free Full Text]

Lin J, Guo J, Gang H, Wojciechowski P, Wigle JT, and Zhang S (2005) Intracellular K⁺ is required for the inactivation-induced high affinity binding of cisapride to HERG channels. Mol Pharmacol 68: 855-865.[Abstract/Free Full Text]

McDonald TV, Yu ZH, Ming Z, Palma E, Meyers MB, Wang KW, Goldstein SAN, and Fishman GI (1997) A MinK-HERG complex regulates the cardiac potassium current I_Kr. Nature (Lond) 388: 289-292.[CrossRef][Medline]

Mitcheson JS, Chen J, Lin M, Culberson C, and Sanguinetti MC (2000) A Structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci USA 97: 12329-12333.[Abstract/Free Full Text]

Mittleman RE and Wetli CV (1984) Death caused by recreational cocaine use. an update. J Am Med Assoc 252: 1889-1893.[Abstract/Free Full Text]

O'Leary ME (2001) Inhibition of human ether-á-go-go potassium channels by cocaine. Mol Pharmacol 59: 269-277.[Abstract/Free Full Text]

Perera R, Kraebber A, and Schwartz MJ (1997) Prolonged QT interval and cocaine use. J Electrocardiol 30: 337-339.[CrossRef][Medline]

Perry M, de Groot MJ, Helliwell R, Leishman D, Tristani-Firouzi M, Sanguinetti MC, and Mitcheson J (2004) Structural determinants of HERG channel block by clofilium and ibutilide. Mol Pharmacol 66: 240-249.[Abstract/Free Full Text]

Riaz K and McCullough PA (2003) Fatal case of delayed repolarization due to cocaine abuse and global ischemia. Rev Cardiovasc Med 4: 47-53.[CrossRef][Medline]

Sanguinetti MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL, and Keating MT (1996) Coassembly of K_vLQT1 and MinK (IsK) Proteins to form cardiac I_Ks potassium channel. Nature (Lond) 384: 80-83.[CrossRef][Medline]

Sanguinetti MC, Jiang C, Curran ME, and Keating MT (1995) A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I_Kr potassium channel. Cell 81: 299-307.[CrossRef][Medline]

Schrem SS, Belsky P, Schwartzman D, and Slater W (1990) Cocaine-induced torsades de pointes in a patient with the idiopathic long QT syndrome. Am Heart J 120: 980-984.[CrossRef][Medline]

Smith PL, Baukrowitz T, and Yellen G (1996) The inward rectification mechanism of the HERG cardiac potassium channel. Nature (Lond) 379: 833-836.[CrossRef][Medline]

Snyders DJ and Chaudhary A (1996) High affinity open channel block by dofetilide of HERG expressed in a human cell line. Mol Pharmacol 49: 949-955.[Abstract]

Spector PS, Curran ME, Zou AR, and Sanguinetti MC (1996) fast inactivation causes rectification of the I_Kr channel. J Gen Physiol 107: 611-619.[Abstract/Free Full Text]

Suessbrich H, Schonherr R, Heinemann SH, Lang F, and Busch AE (1997) Specific block of cloned Herg channels by clofilium and its tertiary analog LY97241. FEBS Lett 414: 435-438.[CrossRef][Medline]

Trudeau MC, Warmke JW, Ganetzky B, and Robertson GA (1995) HERG, a human inward rectifier in the voltage-gated potassium channel family. Science (Wash DC) 269: 92-95.[Abstract/Free Full Text]

Wang S, Morales MJ, Liu S, Strauss HC, and Rasmusson RL (1997) Modulation of HERG affinity for E-4031 by K and C-type inactivation. FEBS Lett 417: 43-47.[CrossRef][Medline]

Weerapura M, Nattel S, Chartier D, Caballero R, and Hebert TE (2002) A comparison of currents carried by HERG, with and without coexpression of MiRP1 and the native rapid delayed rectifier current. is mirp1 the missing link? J Physiol (Lond) 540: 15-27.[Abstract/Free Full Text]

Yang T and Roden DM (1996) Extracellular potassium modulation of drug block of I_Kr. implications for torsade de pointes and reverse use-dependence. Circulation 93: 407-411.[Abstract/Free Full Text]

Zhang S (2006) Isolation and characterization of I_Kr in cardiac myocytes by Cs⁺ permeation. Am J Physiol 290: H1038-H1049.

Zhang S, Kehl SJ, and Fedida D (2003) Modulation of human ether-á-go-go-related K⁺ (HERG) channel inactivation by Cs⁺ and K⁺. J Physiol (Lond) 548: 691-702.[Abstract/Free Full Text]

Zhang S, Rajamani S, Chen Y, Gong Q, Rong Y, Zhou Z, Ruoho A, and January CT (2001) Cocaine blocks HERG, but not KvLQT1+MinK, potassium channels. Mol Pharmacol 59: 1069-1076.[Abstract/Free Full Text]

Zhang S, Zhou Z, Gong Q, Makielski JC, and January CT (1999) Mechanism of block and identification of the verapamil binding domain to HERG potassium channels. Circ Res 84: 989-998.[Abstract/Free Full Text]

Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA, and January CT (1998) Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J 74: 230-241.[Medline]

This article has been cited by other articles:

				M. J. Perrin, P. W. Kuchel, T. J. Campbell, and J. I. Vandenberg Drug Binding to the Inactivated State Is Necessary but Not Sufficient for High-Affinity Binding to Human Ether-a-go-go-Related Gene Channels Mol. Pharmacol., November 1, 2008; 74(5): 1443 - 1452. [Abstract] [Full Text] [PDF]

				H. Gang and S. Zhang Na+ Permeation and Block of hERG Potassium Channels J. Gen. Physiol., June 26, 2006; 128(1): 55 - 71. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.098103v1 317/2/865    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, J. Articles by Zhang, S. Search for Related Content PubMed PubMed Citation Articles by Guo, J. Articles by Zhang, S.