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. 1999 Feb 1;514(Pt 3):667–675. doi: 10.1111/j.1469-7793.1999.667ad.x

Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes

M C Sanguinetti 1, Q P Xu 1
PMCID: PMC2269111  PMID: 9882738

Abstract

  1. The structural basis for the activation gate of voltage-dependent K+ channels is not known, but indirect evidence has implicated the S4-S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4-S5 linker of HERG (human ether-á-go-go-related gene), a human delayed rectifier K+ channel, in Xenopus oocytes.

  2. Mutation of acidic residues (D540, E544) in the S4-S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild-type HERG channels.

  3. Mutation of residues in the S4-S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation

  4. In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization-induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials.

  5. The results indicate that the S4-S5 linker is a crucial component of the activation gate of HERG channels.

Voltage-sensitive K+ channels open in response to membrane depolarization and permit the transmembrane movement of K+ in proportion to its electrochemical driving force. These channels are formed from the co-assembly of four subunits that each contain six (S1-S6) transmembrane domains. Transition of a channel from a non-conducting closed state to a conductive open state in response to membrane depolarization is preceded by outward movement of the positively charged S4 transmembrane domain (Papazian et al. 1991; Aggarwal & MacKinnon, 1996; Larsson et al. 1996) that produces the bulk of the on-gating current (Stühmer et al. 1991; Perozo et al. 1992). The reversal of this process by membrane hyperpolarization produces an off-gating current and channel closure (deactivation). It is not known what couples the movement of the S4 domain to channel opening. However, it is likely that movement of this domain is physically linked to movement of a ‘gate’ that otherwise occludes the channel pore in a closed state. Mutations in the intracellular region that links transmembrane domains S4 and S5, the S4-S5 linker, of Shaker K+ channels alter ion selectivity and single channel conductance (Slesinger et al. 1993). Based on these and other findings, Durell & Guy (1992) proposed that the S4-S5 linker is the activation gate of voltage-activated K+ channels. More recently, it was reported that the activation gating properties of chimeric channels made from Kv2.1 and Kv3.1 K+ channels are determined by the S4-S5 linker (Shieh et al. 1997). In addition, state-dependent modification of residues in the S4-S5 linker of Shaker K+ channels by thiol reactive chemicals suggests that this region moves in concert with the S4 transmembrane domain (Holmgren et al. 1996).

HERG (human ether-á-go-go-related gene) channel subunits co-assemble to form channels that conduct a delayed rectifier K+ current (IKr) important for repolarization of cardiac myocytes (Sanguinetti et al. 1995; Trudeau et al. 1995). To further explore the role of the S4-S5 linker in the activation of K+ channels, we studied the functional effects of charge-altering mutations in this region of HERG (Warmke & Ganetzky, 1994) channels expressed in Xenopus oocytes.

METHODS

Isolation and maintenance of Xenopus oocytes

Oocytes were isolated by dissection from adult Xenopus laevis. The frogs were anaesthetized by immersion in 0.2 % tricaine for 15-20 min, then placed on ice during dissection and removal of ovarian lobes. The incision was sutured closed and the frogs allowed to recover for about 1 month before removal of a second set of oocytes. Frogs were killed by pithing after anaesthetization with tricaine. Clusters of oocytes were digested with 2 mg ml−1 Type 1A collagenase (Sigma) in a Ca2+-free ND96 solution which contained (mM): 96 NaCl, 2 KCl, 2 MgCl2, 5 Hepes (pH 7.6). The isolated oocytes were then incubated at 19°C in Barth's solution containing (mM): 88 NaCl, 1 KCl, 0.4 CaCl2, 0.33 Ca(NO3)2, 1 MgSO4, 2.4 NaHCO3, 10 Hepes (pH 7.4).

Site-directed mutagenesis

Wild-type HERG subcloned into pSP64 vector was prepared as described (Sanguinetti et al. 1995). Site-directed mutagenesis was performed using the megaprimer method described by Sarkar & Sommer (1990). Mutation constructs were confirmed by restriction enzyme and DNA sequence analyses. Complementary RNAs (cRNAs) for injection into oocytes were prepared with SP6 Cap-Scribe (Boehringer Mannheim) following linearization of the expression construct with EcoRI.

Electrophysiology

Methods for cRNA injection and two-electrode voltage clamp of Xenopus oocytes have been described (Goldin, 1991; Goldin & Sumikawa, 1992; Sanguinetti et al. 1995). The external solution was ND96, containing 0.1 mM CaCl2 (22-25°C). pCLAMP software (Axon Instruments) was used to generate voltage-clamp commands, acquire membrane currents, and analyse digitized data.

The voltage dependence of current activation was assessed using standard tail current analysis. Isochronal relationships were determined to avoid K+ accumulation associated with long pulses needed to achieve steady-state activation. Tail currents (I(t)) were fitted with a bi-exponential function:

graphic file with name tjp0514-0667-m1.jpg (1)

which was extrapolated back to the moment of membrane repolarization to determine amplitude (I0). The resulting data were normalized to the maximum current value (Imax) and fitted with a Boltzmann function to obtain relative conductance, Grel:

graphic file with name tjp0514-0667-m2.jpg (2)

where I/Imax is the relative tail current, Vt is the test potential, V0.5 is the half-activation voltage, z is the effective valency, F is Faraday's constant, R is the universal gas constant and T is the absolute temperature. The sign of the exponent term in the Boltzmann function was reversed to fit the voltage dependence of hyperpolarization-activated D540K HERG current (Fig. 6).

Figure 6. Depolarization-activated D540K HERG currents.

Figure 6

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A, currents in response to 1 s voltage pulses from -90 to -40 mV, applied in 10 mV steps from a holding potential of -100 mV. B, currents in response to 1 s pulses from -20 to +20 mV in the same oocyte. C, voltage dependence of activation for D540K and D540A HERG channels. Smooth curves represent best fits of the data to a Boltzmann function. For D540A HERG (0.5 s pulses, n = 17) and D540K HERG (0.5 s pulses, for data between -40 and +60 mV; n = 9), the best fit parameter for V0.5 was -9 mV and for z was 1.4. The data for WT HERG are the same as in Fig. 3B.

The rates of inactivation and recovery from inactivation of wild-type (WT) and mutant HERG channel currents were determined using a three pulse protocol (Spector et al. 1996). An initial 1 s pulse to +40 mV from a holding potential of -80 mV was used to fully activate and/or inactivate channels. A second pulse was applied to -130 mV to allow sufficient time for channels to recover from inactivation, but brief enough to prevent significant deactivation. This requirement was met by using a 25 ms pulse for WT HERG and a 10 ms pulse for the mutant HERG channels. Finally, a third pulse to 0 mV was used to induce re-inactivation of current. The current during this third pulse was fitted with a single exponential function to determine the onset rate for fast inactivation. The rate of recovery from fast inactivation was assessed at -40 to -120 mV after a 400 ms prepulse to 0 mV. The resulting tail current had a rapid rising phase followed by a much slower decay phase (deactivation), and was fitted with a bi-exponential function. The fast component was used as an estimate of the kinetics of recovery from inactivation as described previously (Sanguinetti et al. 1995). Data are expressed as means ±s.e.m. (n = number of oocytes).

RESULTS

Mutation of S4-S5 linker alters kinetics of activation and deactivation

The sequence of the S4-S5 linker of HERG is shown in Fig. 1. This region contains three charged amino acids, D540, R541 and E544. The two acidic residues were individually mutated to Ala to determine if charge neutralization altered the properties of channel activation. Activation of wild-type (WT) HERG channel current in response to membrane depolarization is slow (Fig. 2A) and best described by a bi-exponential function. For example, at -20 mV WT HERG current activates with time constants of 236 ± 7 ms and 1131 ± 42 ms (n = 10). The rate of activation increased, and the current amplitude decreased during 4 s depolarizations positive to -20 mV (Fig. 2E, •). The rectification of the current-voltage relationship is caused by channel inactivation that occurs at a rate faster than channel activation (Shibasaki, 1987; Sanguinetti & Jurkiewicz, 1990; Sanguinetti et al. 1995). Current induced by injection of oocytes with cRNA encoding D540A HERG or E544A HERG activated much faster than WT HERG current. D540A HERG channel current had an initial instantaneous component followed by a bi-exponential activation phase that was about 8 times more rapid than WT HERG (Fig. 2B). E544A HERG current activated more slowly than D540A HERG current, but about 2 times faster than WT HERG current (Fig. 2D). In contrast, mutation of E544 to a basic amino acid (Lys) had an opposite effect on the rate of channel activation. E544K HERG current activated about 2 times slower than WT HERG current (Fig. 2C). Similar to WT HERG, the current-voltage relationships for mutant channels exhibited significant rectification (Fig. 2E). The effects of S4-S5 linker mutations on the rate of HERG current activation at a test potential of -20 mV are summarized in Fig. 3A.

Figure 1. Amino acid sequence of the S4-S5 linker of HERG.

Figure 1

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Figure 2. Depolarization-activated currents in oocytes expressing wild-type or mutant HERG channels.

Figure 2

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A-D, wild-type (WT) and mutant HERG channel currents in response to voltage pulses from -50 to -20 mV applied in 10 mV steps from a holding potential of -80 mV. Tail currents were induced by repolarization to -70 mV. WT HERG and E544K HERG currents were activated with 4 s test pulses; D540A HERG and E544A HERG currents were activated with 1 s pulses. E, current-voltage relationships for the same oocytes shown in A-D.

Figure 3. S4-S5 linker mutations alter HERG current activation.

Figure 3

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A, time constants of current activation at -20 mV (n = 9-17). B, voltage dependence of WT and mutant HERG channel activation. Curves are best fits to a single (WT, E544A HERG) or sum of two (E544K HERG) Boltzmann functions to determine effective valency (z) and half-point (V0.5) of the relationship. WT HERG (4 s pulses, n = 10): V0.5 = -36 mV, z = 3.6; E544A HERG (1 s pulses, n = 13): V0.5 = -31 mV, z = 2.6; E544K HERG (4 s pulses, n = 10): V0.5 = -30 mV, z = 4.2, V0.5′ = -1 mV, z′ = 2.0. Grel, relative conductance.

Mutation of residues in the S4-S5 region also had modest effects on the voltage dependence of channel opening. The voltage dependence of activation for HERG was determined by plotting the relative amplitude of tail currents recorded at -70 mV as a function of test potential. Isochronal (4 s) activation curves were determined for WT and E544K HERG channels because these currents activated very slowly during pulses to potentials < -10 mV. Thus, the V0.5 and slope factor for these channels were underestimated. Activation for D540A and E544A HERG currents was very rapid, and therefore steady-state activation curves could be approximated using 0.5 s test pulses. A Boltzmann function was fitted to the relative tail current amplitudes to obtain the half-point (V0.5) and equivalent charge valency (z). The voltage dependence of activation for E544A HERG using 1 s pulses was only slightly different from WT HERG determined using 4 s pulses (Fig. 3B). The V0.5 and slope factor for E544K and WT HERG cannot be directly compared due to incomplete activation at negative test potentials.

Deactivation of WT HERG current was slow (Fig. 4A) and best described by a bi-exponential function. For example, at -70 mV, the time constants (τd) of WT HERG deactivation were 0.55 ± 0.11 and 1.97 ± 0.17 s (n = 10). Deactivation of E544A HERG and E544K HERG currents were also bi-exponential, but 4-7 times faster than WT HERG. For E544A HERG, τd was 135 ± 2 and 441 ± 18 ms at -70 mV (n = 11). For E544K HERG, τd was 59 ± 2 and 294 ± 13 ms (n = 10) at -70 mV. Deactivation of D540A HERG (Fig. 4B) and D540K HERG currents was mono-exponential and τd was 10 times more rapid than the fast component of WT HERG deactivation. At -70 mV, τd was 49 ± 1 ms for D540A HERG (n = 7) and 60 ± 2 ms for D540K HERG (n = 12). The effects of S4-S5 linker mutations on τd of HERG current at potentials ranging from -120 to -40 mV are summarized in Fig. 4C and D. The altered kinetics caused by single amino acid mutations in the S4-S5 linker of HERG subunits indicate the importance of this region in gating associated with HERG channel activation and deactivation.

Figure 4. S4-S5 linker mutations accelerate the rate of HERG current deactivation.

Figure 4

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A, tail currents recorded from an oocyte injected with WT HERG cRNA. The 2 μA calibration bar refers to these currents. B, tail currents recorded from an oocyte injected with D540A HERG cRNA. The 1 μA calibration bar refers to these currents. C and D, time constants of fast and slow deactivation (n = 9-17).

Mutation of S4-S5 linker has only minor effects on the rate of HERG channel inactivation

Previous studies have shown that rectification of HERG is caused by rapid inactivation that is independent of, and occurs at more negative potentials than, channel opening (Smith et al. 1996; Spector et al. 1996; Wang et al. 1997). Mutations that alter the kinetics of activation would not necessarily be expected to alter the kinetics of inactivation. Therefore, we compared the rates of inactivation for WT and mutant HERG channel currents. The time constant for the onset of inactivation was determined at potentials ranging from +40 to -30 mV, and the recovery from inactivation of HERG was determined at potentials ranging from -40 to -120 mV. An example of the voltage protocol and currents used to measure the onset of, and recovery from, inactivation is illustrated for E544K HERG in Fig. 5A and B, respectively. Compared with the effects on activation and deactivation, point mutations of the S4-S5 linker had only a modest effect on the rates of HERG inactivation (Fig. 5C). For example, at 0 mV the largest changes were a 30 % increase (D540K) and a 27 % decrease (E544K) in the rate of fast inactivation. These findings are consistent with the proposal that activation and inactivation of HERG channels are not coupled (Zou et al. 1998) and that inactivation of HERG involves the S5-S6 loop which lines the outer mouth of the pore (Smith et al. 1996), a region that is distant from the S4-S5 linker.

Figure 5. S4-S5 linker mutations have only minor effects on the rate of HERG current inactivation.

Figure 5

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A, onset of fast inactivation (for oocyte expressing E544K HERG in this example) was determined from single exponential fits of currents during the third pulse of a triple pulse protocol. Fitted traces are superimposed over current traces. Cells were pulsed to +40 mV for 1 s, then hyperpolarized to -130 mV for 10 ms before application of the test pulse to a voltage (Vt) that was varied from +40 to -30 mV. B, recovery from fast inactivation was determined from two exponential fits of tail currents recorded at a voltage (Vt) that was varied from -120 to -40 mV in 10 mV increments following a 0.4 s conditioning pulse to 0 mV. Fitted traces are superimposed over current traces. The fast exponential component was assumed to represent recovery from inactivation, the slow component described the time course of deactivation. C, time constants for the onset of (Vt: -30 to +40 mV) and recovery from (Vt: -120 to -40 mV) fast inactivation in WT and mutant HERG channels (n = 6-12).

Membrane hyperpolarization can reopen D540K HERG channels

Expression of D540K HERG induced a current with unique properties. Outward D540K HERG current activated instantly upon depolarization of the membrane to potentials positive to -100 mV, the reversal potential for a K+-selective channel under the conditions used in these experiments ([K+]o = 2 mM). The current then decayed at a rate that became faster with progressive depolarization (Fig. 6A and B). The voltage dependence of activation of D540K HERG was determined by measuring the amplitude of tail currents at -70 mV after 0.5 s pulses to test potentials ranging from -90 to +60 mV, applied from a holding potential of -100 mV. The relative amplitude of tail currents (Grel) for D540K HERG was nearly identical to D540A HERG for test voltages between -40 and +60 mV. Over this range of voltage, the voltage dependence of both D540A and D540K HERG currents was well described by a Boltzmann function with a V0.5 of -9 mV and a z value of 1.4 (Fig. 6C). For pulses to test potentials between -50 and -90 mV, the tail current amplitude became progressively larger, suggesting the activation of a different open state at negative membrane potentials.

Large hyperpolarizations normally stabilize the closed state of voltage-gated K+ channels, including WT HERG. For example, after a 0.5 s depolarization to +20 mV, repolarization of the membrane to a potential more negative than -110 mV induces a transient inward current. The inward current is due to rapid recovery of channels from inactivation, and is followed by a slower decline due to channel deactivation (Sanguinetti et al. 1995) (Fig. 7B). Similar currents, although with a faster rate of deactivation, were observed for D540A, E544A and E544K HERG (not shown). None of these mutant channels induced slow inward currents upon hyperpolarization. In contrast, D540K HERG current slowly reactivated following deactivation (Fig. 7C). In the absence of a depolarizing prepulse (Fig. 7D), the onset of slow current reactivation had exactly the same time course as reactivation elicited with the prepulse protocol. Thus, reactivation of D540K HERG channels induced by hyperpolarization does not require prior depolarization-induced channel opening. The rate of current reactivation during a 10 s pulse to -150 mV was fitted to a single exponential function. The best fit for the time constant was 1.31 ± 0.84 s (n = 8). These data suggest that D540K HERG channels can occupy an open state (Oh) at very hyperpolarized potentials. Indeed, when the membrane was held at potentials between -100 and -150 mV, the negative holding current increased in proportion to potential (not shown). The magnitudes of instantaneous currents elicited by modest depolarizations (Fig. 6A) are proportional to the chemical driving force for K+ conductance, Vt - EK, through these open channels, followed by a voltage-dependent transition to a closed state (Oh→ C1). Depolarization also permits channels to undergo a voltage-dependent transition to the normal open (O4) and inactivated state (I5) by the usual route of channel gating (Wang et al. 1997) for HERG:

Scheme 1.

Scheme 1

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According to this gating model, the relative amplitude of tail currents is a biphasic function of voltage (Fig. 7C) because of overlap of the voltage dependence for the Oh⇌ C1 and the C3⇌ O4 relationships.

Figure 7. Hyperpolarization causes slow reactivation of D540K HERG channels, but not WT HERG channels.

Figure 7

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A, voltage pulse protocol used to test for hyperpolarization-induced channel reactivation. B, currents in an oocyte expressing WT HERG. Membrane repolarization induced an initial rapid inward current (recovery from inactivation), followed by a decay phase to the zero current level (deactivation). C, currents in an oocyte expressing D540K HERG. Membrane repolarization induced an initial decaying inward current similar to WT HERG. Unlike WT HERG, current slowly increased (reactivated) after the initial decay phase. The slow inward current became increasingly larger as the return potential was made more negative from -110 to -150 mV. D, reactivation of D540K HERG channel current induced by hyperpolarization from a holding potential of -100 mV without a prior depolarization. Currents were recorded from the same oocyte used to record the currents shown in C.

The rate of decay of hyperpolarization-activated D540K HERG current upon depolarization (Oh→ C1) was bi-exponential and varied as a function of voltage (not shown). The time constants for current decay were 13.5 ± 0.8 and 657 ± 93 ms at -90 mV, and 35 ± 1.4 ms and 1.62 ± 0.16 s at -40 mV (n = 7). The voltage dependence of the Oh→ C1 transition for D540K HERG was determined using a dual-pulse protocol where the extent of channel closure was estimated from a plot of the relative amplitude of peak current during a pulse to 0 mV versus the voltage of a preceding 10 s conditioning pulse (Fig. 8A and B). This relationship was fitted with a Boltzmann function with a V0.5 of -117 mV (Fig. 8C), 40 mV more negative than the V0.5 for fast inactivation of WT HERG current (Zou et al. 1998).

Figure 8. Voltage-dependent availability of the hyperpolarization-induced reactivated state of D540K HERG channels.

Figure 8

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A, currents recorded during a 10 s conditioning voltage pulse applied between -170 and -40 mV in 10 mV steps, followed by a common test pulse to 0 mV to assay channel availability. B, expanded view of currents outlined by the dashed box in A. C, relative amplitude of peak currents shown in B. The smooth curve represents the best fit of the data to a Boltzmann function having a V0.5 of -117 mV and a z of 2.6 (n = 8).

The gating of D540K HERG, with distinct open states activated by either depolarization or hyperpolarization is unlike any other previously described channel. How might hyperpolarization cause re-opening of the D540K HERG channel? We considered the possibility that inward flow of K+ through the channel at potentials negative to EK might cause electrostatic repulsion of K540 and cause channel opening. However, as shown in Fig. 9, removal of extracellular KCl did not prevent the channel from opening upon hyperpolarization. Replacement of extracellular KCl with NaCl prevented the flow of inward current upon hyperpolarization to -150 mV. However, subsequent repolarization to -90 mV still induced a decaying outward tail current that resulted from deactivation of channels that were opened by the prior hyperpolarization (n = 3). The outward current was larger in 0 mM than in 2 mM extracellular K+ because of the increased chemical gradient. Thus, the opening of D540K HERG channels induced by hyperpolarization is not caused by electrostatic repulsion between K+ and the positively charged Lys at position 540.

Figure 9. D540K HERG channels reactivate upon hyperpolarization even in the absence of extracellular K+.

Figure 9

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Hyperpolarization to -150 mV (4 s duration) activated an inward current in an oocyte bathed in 2 mM K+ ND96 solution (2 K+). Repolarization to -90 mV activated an outward current that decayed over a few seconds, reflecting closure of channels opened during the pulse to -150 mV. The solution was then changed to K+-free ND96 solution (0 K+) and the pulse protocol repeated. In the absence of extracellular K+, no current (except leak) was activated upon hyperpolarization to -150 mV, but repolarization to -90 mV activated a large outward current, indicating that channels were open at -150 mV.

DISCUSSION

Previous studies have demonstrated that the S4-S5 linker is the putative docking site for the inactivation particle (gate) of both K+ and Na+ channels. In Shaker K+ channels, the N-terminus of each subunit can act as an inactivation gate (Hoshi et al. 1990). This region binds to a site on the cytoplasmic S4-S5 linker when the channel is in the open state (Isacoff et al. 1991). In the Na+ channel, the intracellular loop between domains III and IV appears to bind to the S4-S5 linker of domain III to inactivate the channel. For example, fast inactivation of the rat brain Na+ channel can be disrupted by mutation of a single amino acid, A1329) of the S4-S5 linker (Smith & Goldin, 1997). The present study demonstrates that the S4-S5 linker is also important in the activation gating of HERG K+ channels.

Mutation of charged residues in the S4-S5 linker of HERG K+ channels altered the rate of current activation and deactivation, while having only minor effects on fast inactivation or the voltage dependence of activation. These findings indicate that the S4-S5 linker somehow modulates channel opening and closing. D540K HERG channels had the unique property of hyperpolarization-induced re-opening. The voltage dependence of D540K HERG activation was identical to D540A HERG when assessed by depolarization to potentials positive to -40 mV. This indicates that the voltage-sensing properties of D540K HERG channels in response to membrane depolarization are relatively normal. However, membrane hyperpolarization induced channel re-opening, instead of a stabilization of the closed state of the channel. The unique importance of D540 in stabilization of HERG channels in a closed state is indicated by the finding that mutation of a nearby negatively charged residue to Lys (E544K) did not share with D540K the property of channel hyperpolarization-induced channel re-opening. At negative transmembrane voltages, D540 of wild-type HERG may form a salt bridge with a positively charged residue on a nearby domain of the same or adjacent subunit to stabilize the closed state of the channel. Substitution of D540 with a positively charged Lys may result in repulsion of the linker away from its hyperpolarization-favoured position that normally occludes the inner vestibule of the pore.

There is no direct evidence that the S4-S5 linker occludes the inner pore in the closed state, or that outward movement of the S4 region moves the S4-S5 linker. However, previous experimental findings are consistent with such a model. First, the docking site on the S4-S5 linker for the N-terminal ball of Shaker K+ channels is only accessible when the channel is in the open state (Isacoff et al. 1991). This suggests that channel opening must result in movement of the linker into a position that unmasks the N-terminus binding site. Second, deactivation can trap drug molecules in the inner vestibule of the channel pore of Shaker K+ channels. This finding suggests that the putative activation gate behaves like a trapdoor (Holmgren et al. 1997).

Mutations in the S4-S5 linker significantly increased the rate of HERG channel deactivation. Previous studies demonstrated that deletion of the N-terminus of HERG greatly accelerated the rate of channel deactivation (Schönherr & Heinemann, 1996; Spector et al. 1996). The N-terminus of HERG may interact with the S4-S5 linker to affect these changes (Wang et al. 1998). The N-terminus of eag K+ channels also modulates gating. Deletion of amino acids 7-12 of the N-terminus of eag channels greatly slowed the rate of deactivation, an effect that can be reversed by a single mutation (H343R) in the putative S4 transmembrane region (Terlau et al. 1997). Thus, binding of the N-terminus of HERG (or eag) to the S4-S5 linker (or S4 transmembrane region) modulates channel deactivation but does not promote fast inactivation as it does in Shaker and several other voltage-dependent K+ channels. Thus, the S4-S5 linker could potentially be both the N-terminal ball docking site and the activation gate of HERG K+ channels.

In summary, our findings indicate a critical role for the S4-S5 linker in gating of the HERG K+ channel. At negative transmembrane potentials the linker may occlude the inner vestibule of the pore and stabilize the channel in a closed state. Outward movement of the S4 region induced by membrane depolarization may drag the S4-S5 linker with it and allow the channel to open. The S4-S5 linker is not highly conserved among the different families of voltage-gated K+ channels. Nonetheless, this region also modulates the activation gating of Kv2.1 and Kv3.1 K+ channels (Shieh et al. 1997) and may serve a similar role in other voltage-dependent channels.

Acknowledgments

We thank M. Lin for technical assistance and Drs M. Tristani-Firouzi and O. McManus for comments on the manuscript. This work was supported by the National Heart Lung and Blood Institute of the NIH (HL55236) and a Grant-in-Aid Award from the American Heart Association and Pfizer, Inc.

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