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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales; 2St. Vincent's Clinical School, University of New South Wales, New South Wales, Australia; 3Department of Computer Science, University of Wisconsin at River Falls, River Falls, Wisconsin; and 4Department of Physiology, University of Cambridge, Cambridge, United Kingdom
Submitted 30 November 2005 ; accepted in final form 30 January 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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potassium channel; kinetics; voltage-dependent gating
hERG K+ channels resemble other voltage-gated K+ channels in containing six transmembrane domains (S1–S6) with a pore helix between the S5 and S6 domains and a primary activation voltage sensor in the positively charged S4 transmembrane domain (54, 59). Yet, hERG K+ channels show an unusual combination of slow activation and deactivation with rapid and voltage-dependent inactivation and recovery from inactivation (46, 47, 49, 51). hERG K+ channel inactivation resembles the C ("collapse of pore")-type inactivation seen in Shaker K+ channels (3, 21) in its sensitivity to mutations in the outer pore region (11, 25, 47, 49), externally applied tetraethylammonium (11, 49), and increased extracellular K+ concentration (11, 49, 57). However, inactivation in hERG is distinct in its marked voltage sensitivity and rapid kinetics (47, 49, 51), and these kinetic features in turn confer the apparent inward rectification that minimizes current flow through hERG K+ channels at depolarized potentials but maximizes it during terminal repolarization of the cardiac action potential (14, 27, 61). The latter properties are critical both for normal cardiac repolarization and suppression of arrhythmias initiated by premature excitation (27, 49).
There has been considerable progress in identifying the molecular regions and residues crucial for the gating properties of hERG [e.g., activation (37, 38, 50, 52, 60), deactivation (26, 33, 58), and inactivation (12, 15, 25, 47, 49, 53)]. However, our understanding of the physical basis and the energetics of hERG gating are less well developed. Temperature has often been used to probe the energetics of conformational changes in proteins, including ion channels (8, 24, 29, 30, 41–43). Here, we studied the temperature dependences of activation and inactivation in hERG K+ channels. Our findings provide an explanation for the marked temperature dependence of hERG currents during cardiac action potential waveforms. Furthermore, our data indicate that the energy barrier between the open and inactive states must be composed of a significant enthalpic barrier and a positive entropic component.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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48 h after being plated on microscope coverslips. Cells were superfused with normal Tyrode solution containing (in mM): 130 NaCl, 4.8 KCl, 0.3 NaH2PO4, 0.3 KH2PO4, 1 MgCl2, 1 CaCl2, 12.5 glucose, and 10 HEPES (titrated to pH 7.4 with NaOH). The temperature at the cell was maintained by either preheating the perfusion solution and by heating the microscope oil immersion lens with a water jacket as previously described (27) or by heating/cooling the perfusion solution using a TC2bip Bipolar temperature controller (Cell MicroControls, Wellesley Hills, MA). In the latter experiments the thermocouple monitoring temperature was placed in the perfusion chamber, and patched cells were lifted off the chamber bottom.
Patch Clamping
Borosilicate glass tubing (Clark Electromedical, Reading, UK) patch pipettes, with resistances of 2–4 M
when filled with internal solution, were pulled using a horizontal puller (Sutter P87) and fire polished. The internal solution contained (in mM) 120 potassium gluconate, 20 KCl, 1.5 MgATP, 5 EGTA, and 10 HEPES (pH 7.3 with KOH). The liquid-liquid junction potential between the internal and external solution, calculated to be –15 mV (2), has been corrected for in all experiments. Currents were amplified (Axopatch 200A or Axopatch 200B amplifier; Axon Instruments, Foster City, CA) and digitized (Digidata 1322; Axon Instruments) before storage on a personal computer. Capacitance current transients were electronically subtracted, and series resistance compensation was typically 80%. Current signals were digitized and low pass filtered at 2 and 1 kHz, respectively (for assessment of steady-state activation), 5 and 2 kHz, respectively (for rates of activation), and 25 and 10 kHz, respectively (for rates of inactivation and recovery from inactivation as well as steady-state inactivation). All current traces were leak subtracted off-line, assuming a linear leak in the range –150 to +60 mV. Acquisition and analysis of data were performed using pClamp software (Axon Instruments).
Voltage Protocols Whole cell conductance. Whole cell conductances of fully activated channels were measured at test potentials in the range +40 to –120 mV in cells exposed to temperature changes in the range 22–37°C. Cells were depolarized from a holding potential of –80 to +40 mV for 1 s to fully activate and inactivate the channels, followed by a 10-ms step to –140 mV to allow channels to recover from inactivation, and then stepped to voltages in the range +40 to –120 mV. Finally, the voltage was stepped to –140 mV for 500 ms. Single exponential functions were fitted to the –140-mV tail current recordings to estimate the rate of deactivation in each experiment. The measured peak currents at each potential were corrected for the channel deactivation that occurred during the 10-ms step to –140 mV. Similar to that reported by Smith et al. (49), we found that currents measured using this protocol showed a linear response to voltage.
The temperature sensitivity of the whole cell conductance for fully activated channels and reaction rates (see below) were calculated as Q10 values defined as the change in rate (or conductance) for each 10°C change in temperature (16)
 | (1) |
1 and 2 are the rate constants or conductances, measured at temperatures T1 and T2.
Voltage dependence of activation.
The voltage dependence of activation was measured using a standard isochronal tail current protocol (52, 57). Cells at a holding potential of –80 mV were subject to 30-s depolarizing steps to voltages in the range –80 to +40 mV before stepping the voltage to –120 mV where tail currents were recorded. Depolarization steps of 30 s were used because hERG channels activate very slowly at small depolarizations. The resulting isochronal activation curves provide information about the distribution between the groups of closed and open states amenable to analysis using a simple Boltzmann function:
 | (2) |
 | (3) |
G0 is the difference in Gibbs' free energy between states at 0 mV, zg is the effective number of electronic charges traversing the membrane electric field during activation, F is Faraday's constant, R the universal gas constant, and T the absolute temperature. G0 is also often referred to as the chemical component and zgFVm as the electrostatic component of the free energy difference between the two states.
The simplified Boltzmann function analysis of steady-state activation described above may lead to a significant underestimation of the real value of gating charge, zg, moved during opening of the channels (1). A more accurate estimate can be obtained from measurement of the limiting (lim) slope of the logarithm of the open probability (1):
 | (4) |
Fig. 2, inset).
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Fig. 5, inset). During this second voltage step, channels rapidly reequilibrate into a new open-inactive state distribution (depending on voltage) and then start to deactivate at potentials less than approximately –20 mV. The peak amplitude of the tail current at each potential, after recovery from inactivation, was corrected for channel deactivation (as described in Ref. 49), and these current values were converted to conductances (by dividing by the electrochemical driving force). Strictly speaking, this protocol really measures the voltage dependence of the recovery from inactivation, although for convenience we refer to the curves as steady-state inactivation curves. Steady-state inactivation curves were analyzed using a similar Boltzmann distribution analysis described above for steady-state activation to provide indications of the distribution between the groups of open states and inactivated states (13, 37).
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 | (5) |
is the latency to activation, 
is the time constant of activation, and t is time. Rates of deactivation. Rates of deactivation were measured from tail currents recorded in the voltage range –120 to –100 mV after a 1-s depolarization step to +40 mV to activate the channels. hERG tail currents at negative voltages show a hooked appearance (46, 49), an initial increase because of recovery from inactivation, followed by a declining phase because of deactivation. Deactivation of hERG K+ channels has been reported to occur via either a single- or double-exponential time course (see discussion in Ref. 52). However, at negative voltages such as those studied here, deactivation occurs predominantly if not exclusively via a monoexponential process (57). In this study, we have reported just the fast component rates that were obtained either by fitting a double-exponential function to both portions of the hooked tail current (recovery from inactivation and deactivation, see Fig. 6) or by fitting a single exponential to the decaying phase of tail currents between the 25 and 75% current levels.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Temperature Sensitivity of hERG K+ Channel Conductance in Whole Cell Recordings
We first investigated whether temperature-dependent changes in hERG K+ channel conductance could explain the effects observed in Fig. 1. Whole cell currents of fully activated channels showed a linear response to voltage (see Fig. 2). In the example shown in Fig. 2, the whole cell conductance, measured from the slope of the I-V plots, increased from 29.3 nS at 20.5°C to 50.2 nS at 33.9°C. The mean value of Q10 for whole cell conductance in the voltage range –120 mV to +40 mV was 1.40 ± 0.03 (n = 7), which agrees closely with previous reports for other ion channels (16). The temperature sensitivity of whole cell conductance therefore accounts for only part (25% in the case of a 3-s step to 0 mV) of the temperature sensitivity of the currents shown in Fig. 1.
Temperature Sensitivity of Steady-state Activation Figure 3A shows a typical family of currents recorded at 14°C in response to 30-s depolarization steps to voltages in the range –80 to +40 mV after which the voltage was stepped to a level of –120 mV. Figure 3B plots the peak tail current amplitudes obtained during the –120-mV step against the voltage of the preceding depolarization step. The line of best fit to a Boltzmann function (Eq. 1 in MATERIALS AND METHODS) in Fig. 3B gave a V1/2 for current activation of –26.8 mV. Mean ± SE data for the voltage dependence of activation at 32, 22, and 14°C are plotted in Fig. 3C. Increased temperature shifted the V1/2 for activation in a negative direction, giving values of –29.6 ± 2.3 (n = 5), –38.7 ± 1.2 (n = 5), and –44.2 ± 2.1 (n = 7) mV at 14, 22, and 32°C, respectively (Fig. 3C). Increased temperature also altered the slopes of the curves in Fig. 3C. The value of the slope factor at 14°C (8.0 ± 0.3 mV) was statistically different from the values at 22°C (5.8 ± 0.6 mV) and 32°C (5.5 ± 0.3 mV). The slope factors at 22 and 32°C were not significantly different from each other. Thus, in Fig. 3C, the decrease in temperature from 32 to 22°C results in a parallel leftward shift of the whole curve, but the further leftward shift seen after the reduction in temperature to 14°C appears to be because of a combination of an altered slope and a shift in the midpoint of the curve.
The use of simple Boltzmann distribution analysis of steady-state activation curves can lead to significant underestimates of the effective number of electronic charges moved during channel activation. A more accurate estimate of the number of electronic charges moved during channel activation can be obtained from the limiting slope (1) of the logarithm of the open probability (approximated as I/Imax) plotted against voltage (Fig. 4A). This analysis reveals a trend with values at 14°C (5.9 ± 0.6 e–) significantly different from those at 22 and 32°C (7.9 ± 1.0 and 8.5 ± 0.7 e–, respectively; Fig. 4B).
Temperature Sensitivity of Steady-State Inactivation Figure 5A shows families of currents recorded from the same cell during protocols to measure the voltage dependence of recovery from inactivation at 32 and 22°C. Cells were depolarized from a –80 mV holding potential to +40 mV for 1 s to allow channels to activate and inactivate. The potential was then stepped to voltages in the range –150 to +60 mV for 500 ms to allow channels to recover from inactivation. For voltages less than –50 mV, the channels will start to deactivate during the 500-ms tail pulse. Deactivation was corrected for (Fig. 5Aii) to ensure measurement of the true peak tail currents (compare Fig. 5Aii and Fig. 5B, as explained in MATERIALS AND METHODS). The latter were converted to conductances (see Fig. 5C), and the data were fitted with a Boltzmann function. Figure 5D plots the voltage dependence of inactivation data (mean ± SE) at 32, 22, and 14°C. Increased temperature shifted the V1/2 of inactivation in a depolarizing direction from –95.6 ± 5.1 mV (n = 4) at 14°C to –88.3 ± 0.7 mV (n = 4) and –78 ± 4.4 mV (n = 3) at 22 and 32°C, respectively, but did not significantly influence the slope factors (14°C: –24.8 ± 2.3 mV; 22°C: –26.5 ± 2.3 mV; 32°C: –25.5 ± 1.2 mV). These data suggest that temperature has a strong effect on the apparent voltage sensitivity of hERG inactivation.
Temperature changes produced opposite effects on activation and inactivation. Thus increasing temperature produced negative shifts in the voltage dependence of activation (more negative G0, in thermodynamics parlance, see Eq. 3 in MATERIALS AND METHODS) but positive shifts in the voltage dependence of inactivation (less negative G0; Table 1). This would result in an overall increase in the voltage range at which channels occupy the open state under steady-state conditions as temperature increases. From the product of the steady-state activation and steady-state inactivation curves, one can calculate the mean steady-state occupancy of the open state at different voltages (see Fig. 6). The voltage for the peak open state occupancy shifts from approximately –22 mV at 14°C to approximately –38 mV at 32°C. From Fig. 6, we can also calculate the mean occupancy at 0 mV, which increases from 2.0% at 14°C to 4.5% at 32°C. This temperature-dependent increase in occupancy of the open state at 0 mV, when combined with the temperature sensitivity of whole cell conductance, can account for the temperature sensitivity of the magnitude of currents recorded under steady-state conditions (see Fig. 1). However, during the cardiac action potential, hERG channels will not reach steady state. Therefore, to fully characterize the temperature dependence of hERG currents, we investigated the effect of temperature on the kinetics of hERG gating.
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Activation Activation of hERG K+ channels involves transitions through multiple preopen closed states (13, 37, 57) and therefore will involve multiple kinetic steps that cannot be isolated in whole cell current recordings. Furthermore, whole cell current recordings in wild-type hERG channels are complicated by the concomitant inactivation of the channels. We can, however, separate the channel activation and inactivation processes using an envelope of tails protocol (57) and thereby obtain an estimate of the rate constant for the slowest step in the activation pathway. Figure 8, A and B, shows typical families of currents recorded from the same cell at 22 and 37°C during an envelope of tails protocol to measure rates of activation at 0 mV. The peak amplitudes of the tail currents, indicated in Fig. 8, A and B, are plotted against test pulse duration in Fig. 8Ci and logarithmic (Fig. 8Cii) time scales. The use of the logarithmic time scale in Fig. 8Cii clearly highlights the latency before channels start to open. This latency reflects the time taken to traverse through the multiple other steps in the activation pathway. The lines of best fit shown in Fig. 8C were obtained by fitting a single exponential to the later half of the data set (see Eq. 5). In the example shown in Fig. 8, the time constant of the exponential function fitted to the last half of the 0 mV data set decreased from 603 ms at 22°C to 171 ms at 37°C. This is equivalent to a Q10 of 2.3.
Inactivation Figure 9A shows typical hERG currents recorded during voltage protocols to monitor the rate of inactivation at +40 mV. Cells were depolarized to +40 mV (for 1 s) from a holding potential of –80 mV, stepped to –80 mV for 20 ms to allow recovery from inactivation and then stepped to +40 mV for 100 ms to monitor inactivation at +40 mV. Inactivation was well fitted with a single exponential function (Fig. 9A, inset). In the example shown in Fig. 9, temperature changes between 24 and 37°C increased rates of inactivation 3.8-fold, corresponding to a Q10 value of 2.8.
Recovery from Inactivation and Deactivation Figure 9B shows typical examples of tail currents recorded at –120 mV from a single cell at 24 and 37°C. They show an initial increase in inward current, with time constant of 1.1 ms at 37°C, that reflects recovery from inactivation. This is followed by a decrease in current, with a time constant of 12.4 ms at 37°C (Fig. 9B, inset), reflecting deactivation. The currents recorded at 24 and 37°C could not be scaled using a single time scale factor: the initial phases of current increase and subsequent decay phases required scaling by time factors of 4 (Fig. 9C) and 2.3 (see Fig. 9D), corresponding to Q10 values of 2.9 and 1.9, respectively.
The processes of activation, inactivation, recovery from inactivation, and deactivation in the hERG K+ channel have differing temperature sensitivities. To a first approximation, the differences in temperature sensitivity can be compared by calculating the Q10 factor, defined as the change in rate (or conductance) for each 10°C change in temperature (see Eq. 1). Figure 10 summarizes the Q10 factors for the rate constants measured in this study. The rates of activation (at +40 mV) and deactivation (at –120 mV) were less temperature sensitive [Q10 factors, 2.1 ± 0.1 and 1.7 ± 0.1 (n = 9), respectively] than the rates of inactivation and recovery from inactivation [Q10 factors, 2.5 ± 0.2 (n = 7) and 2.6 ± 0.1 (n = 7), respectively].
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2.5-fold increase in current for each 10°C rise in temperature during 3-s depolarization steps to 0 mV (Fig. 1). Increased temperatures shifted V1/2 values for steady-state activation in a negative direction (from –29.6 ± 2.3 mV at 14°C to –44.2 ± 2.1 mV at 32°C: Fig. 2C) but V1/2 for inactivation in a positive direction (from –95.6 ± 5.1 mV at 14°C to –78.0 ± 4.4 mV at 32°C; Fig. 4D and Table 1). Despite there being numerous studies of the effects of temperature on the kinetics of ion channel gating, there are relatively few studies on the effect of temperature on steady-state activation and inactivation that we use for comparison with our results. Similar to the results reported here, increased temperature has been reported to result in depolarizing shifts in V1/2 of inactivation for voltage-gated Na+ channels (35, 40, 44, 48) and for C-type inactivation in Shaker K+ channels (29). Conversely, increased temperature results in only small shifts in V1/2 of activation for Shaker K+ channels (43) and minimal shifts for Na+ channels (40, 44, 48). The effects of temperature on the V1/2 of activation of hERG therefore appears to be very different from those reported for Shaker K+ channels and voltage-gated Na+ channels. There are not complete data sets available for other channels. However, in an early study of K+ channels in T lymphocyte (36), it was reported that increased temperature resulted in a depolarizing shift in steady-state inactivation and a hyperpolarizing shift in steady-state activation, similar to that reported here for hERG. Also, studies on slow delayed-rectifier K+ channels (minK + Xenopus KCNQ1; see Ref. 5) show a greater temperature sensitivity of current magnitude at lower voltages compared with higher voltages, consistent with increased temperature, resulting in a hyperpolarizing shift in the steady-state activation of these channels. Thus it is likely that more extensive characterization of other ion channel clones would reveal channels with similar temperature dependencies for steady-state activation and inactivation as that shown here for hERG.
The combination of a depolarizing shift in the V1/2 of steady-state inactivation and hyperpolarizing shift in V1/2 of activation with increases in temperature for hERG K+ channels will result in a large temperature-dependent increase in the voltage range that favors open states (Fig. 6). This is most notable in the range –60 to –20 mV, precisely the voltage range over which hERG currents contribute most to repolarization of the cardiac action potential (14, 27, 61). This property can account for the larger hERG currents observed at higher temperatures during action potential voltage clamps (Fig. 1B). Conversely, decreased temperature should diminish hERG current and prolong ventricular repolarization, as observed during hypothermia (22).
The effect of temperature on the voltage sensitivity of activation and inactivation showed significant differences. In the temperature range 14–32°C, there were no significant changes in the slope of the steady-state inactivation curves (Fig. 4D). Conversely, the slope factor for the voltage dependence of activation decreased significantly between 14 and 22°C but was then steady between 22 and 32°C (Fig. 2C). Similarly, there was significantly less charge moved during activation of hERG K+ channels at 14°C compared with 22 and 32°C (Fig. 3B). Rodriguez et al. (43) also noted a decrease in the total amount of charge moved during activation of Shaker K+ channels at temperatures <10°C when studied in Xenopus oocytes. One possible explanation for these results could be that there is a phase transition in the cell membranes that in mammalian cells occurs at temperatures <22°C (our studies) but at a temperature <10°C in amphibian cells. Given that the ambient temperature experienced by amphibian cells (typically 15–25°C) is 15°C below that experienced by mammalian cells, it is reasonable to assume that the phase transition for amphibian cell membranes would be correspondingly lower than that for mammalian cell membranes (compare, e.g., Refs. 41 and 43 with Ref. 45).
The change in effective charge moved with decreasing temperature has two important implications for hERG K+ channels. First, when investigating the effects of temperature on rates of activation, one has to bear in mind that the process might be quite different at temperatures <22°C compared with those at temperatures >22°C. For this reason, we restricted our analysis of the effects of temperature on rates of activation and deactivation to temperatures >22°C. Second, the absence of a change in slope for inactivation at low temperatures is consistent with the hypothesis that the voltage sensitivity of inactivation is not tightly coupled to the voltage sensitivity of activation (see, e.g., Refs. 17 and 62).
Effects of Temperature on Kinetics of hERG K+ Channel Gating In principle, the power of studying the effect of temperature on ion channel kinetics is that it makes it possible to dissect the enthalpic and entropic components of changes in free energy for each kinetic process (43). However, given that each of these kinetic processes in hERG are likely to be complex multistep processes, it is only possible to obtain estimates of the rate constants for the rate-limiting step in each process. Furthermore, there can be no guarantee that the rate-limiting step in these complex pathways will necessarily be the same at each temperature. Nevertheless, the data obtained in this study provide some useful starting points when thinking about the thermodynamics of hERG gating.
The first and most important conclusion that we can make is that the rates of activation, deactivation, inactivation, and recovery from inactivation all have different temperature sensitivities. This is not surprising, given that each of the gating steps is likely to involve different conformational changes in the channel and so require the breaking and/or forming of chemical bonds with different energies. Furthermore, in previous studies where the temperature sensitivity of individual kinetic steps have been studied, they have invariably been found to be different [see, e.g., Shaker K+ channels (29, 43), voltage-gated Na+ channels (23, 35, 44), and slow delayed-rectifier K+ channels (6)]. The differences in the temperature sensitivities of each of the kinetic processes that govern hERG channel activity mean that it is not possible to use data obtained at room temperature and extrapolate the results to physiological temperatures using a single temperature scaling factor. Rather, if one wants to understand the clinical significance of a disease causing mutation or understand the regulation of the channel under physiological conditions, it is preferable to study the kinetics at 37°C (see, e.g., Ref. 28). It is also possible that the apparent temperature dependence of drug and toxin binding to hERG (10, 20, 31) could be because of the state dependence of drug and toxin binding coupled with a temperature dependence of the occupancy of different conformational states of the channel. Thus, to obtain accurate estimates of the potencies of drugs that inhibit hERG, it would also be preferable to undertake the studies at 37°C.
Second, the rates of inactivation and recovery from inactivation were more sensitive to changes in temperature than were the rates of activation and deactivation. This suggests that there must be a higher enthalpic barrier for inactivation/recovery from inactivation than for activation/deactivation (32). The rates of inactivation and recovery from inactivation (of the order of 1–10 ms), however, are considerably faster than the rates of activation and deactivation (10s-100s of ms). Therefore, the total energy barrier for inactivation and recovery from inactivation must be lower than for activation and deactivation. The total energy barrier, however, is composed of the temperature-sensitive enthalpic component and an entropic component (39); therefore, the energy barriers for inactivation and recovery from inactivation must contain a significant positive entropic component as well. At present, it is not possible to measure specific thermodynamic parameters for the activation/deactivation and inactivation/recovery from inactivation processes for hERG gating, since it is not possible to isolate the individual kinetic steps in the complex pathways involved in each process. This task will be very difficult. However, if we can identify mutations that slow individual kinetic steps, then it should be possible to start to obtain estimates of the thermodynamic parameters for those steps and then build up a complete thermodynamic model of the kinetics of hERG channel gating.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, where 
), 22°C (
), and 14°C (
).
). In ii, broken lines show how current traces were corrected for deactivation to enable estimation of peak tail current. Solid arrows show peak tail currents, and broken arrows show peak tail currents corrected for deactivation. Dotted lines show zero current level. B: plots of measured peak tail currents (
) and peak tail currents corrected for deactivation (
