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Gating current studies reveal both intra- and extracellular cation modulation of K⁺ channel deactivation

Zhuren Wang, Xue Zhang and David Fedida

MS 8575 Received 31 July 1998; accepted after revision 24 November 1998.

	ABSTRACT

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1. The presence of permeant ions can modulate the rate of gating charge return in wild-type human heart K⁺ (hKv1.5) channels. Here we employ gating current measurements in a non-conducting mutant, W472F, of the hKv1.5 channel to investigate how different cations can modulate charge return and whether the actions can be specifically localized at the internal as well as the external mouth of the channel pore.

2. Intracellular cations were effective at accelerating charge return in the sequence Cs⁺ > Rb⁺ > K⁺ > Na⁺ > NMG⁺. Extracellular cations accelerated charge return with the selectivity sequence Cs⁺ > Rb⁺ > Na⁺ = NMG⁺.

3. Intracellular and extracellular cation actions were of relatively low affinity. The K_d for preventing slowing of the time constant of the off-gating current decay (_off) was 20·2 mM for intracellular Cs⁺ (Cs⁺_i) and 358 mM for extracellular Cs⁺ (Cs⁺_o).

4. Both intracellular and extracellular cations can regulate the rate of charge return during deactivation of hKv1.5, but intracellular cations are more effective. We suggest that ion crystal radius is an important determinant of this action, with larger ions preventing slowing more effectively. Important parallels exist with cation-dependent modulation of slow inactivation of ionic currents in this channel. However, further experiments are required to understand the exact relationship between acceleration of charge return and the slowing of inactivation of ionic currents by cations.

	INTRODUCTION

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In voltage-gated K⁺ channels the voltage dependence of the return of gating charge after depolarization is bimodal. When depolarizations are sufficient to cause channel opening, in the absence of permeating ions, on repolarization there is a rising phase of off-gating current and slow subsequent decay (Perozo et al. 1993; Stefani et al. 1994). Some slowing is due to the relative voltage independence of the last closed-open transition (Zagotta & Aldrich, 1990), but we have demonstrated that much of this effect could be prevented by the presence of cations (Chen et al. 1997) and suggested that either cations were able to allosterically modulate K⁺ channel deactivation, or alternatively that the slowing was predominantly caused by an accelerated inactivation that could be partially prevented by the presence of cations (Yellen, 1997). This latter explanation fits with ionic current data showing that C-type inactivation is accelerated when the concentration of extracellular cations is lowered (Levy & Deutsch, 1996), or removed altogether (Baukrowitz & Yellen, 1995; Kukuljan et al. 1995). In the preceding paper (Fedida et al. 1999) we have shown that progressive reduction of intracellular cations is a more potent way to accelerate the process of slow inactivation in Kv1.5.

In our previous study we did not attempt to localize the action of cations to the intracellular or extracellular domains, due to the very nature of the experiments on conducting channels (Chen et al. 1997). The aim of the present study was to examine cation modulation of gating currents in a non-conducting mutant (NCM) channel to try to determine the species, concentration dependence and sidedness of action of cations in the absence of ion conduction. In wild-type channels the sidedness can be difficult to unequivocally demonstrate as outward ionic currents can lead to ion accumulation and alter the milieu at the outer pore mouth, independently of bulk extracellular cation concentration and species, as pointed out by Baukrowitz & Yellen (1995). A potential solution to this problem was to examine cation modulation in the absence of ion conduction. The NCM channel was superior in this regard as ion conduction through the pore could be essentially eliminated.

In these gating current experiments on hKv1.5 we have made parallel observations to those we have described in the preceding work on ionic currents. Gating charge return could be modulated at both intracellular and extracellular low affinity regulatory sites, with a 10-fold higher affinity at the intracellular site (Fedida et al. 1999). We also observed that Cs⁺ was most effective at preventing the slowing and immobilization of off-gating charge. We have discussed our observations in terms of mechanisms by which deactivation can be accelerated by the presence of monovalent cations.

	METHODS

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Cells and solutions

Kv1.5 in the plasmid expression vector, pRC/CMV was mutagenized using the Stratagene Chameleon Kit (La Jolla, CA, USA) such that tryptophan 472 was converted to phenylalanine (W472F). This non-conducting mutation (NCM) is analogous to the ShH4-IR W434F (Perozo et al. 1993). HEK 293 cells were transiently or stably transfected with wild-type (WT) hKv1.5 or W472F-hKv1.5 (NCM) cDNAs in pRC/CMV, using LipofectACE reagent (Canadian Life Technologies, Bramalea, ON, Canada) in a 1:10 (w/v) ratio. Patch pipettes contained (mM): N-methyl-D-glucamine (NMG⁺), 140; EGTA, 5; MgCl₂, 1; Hepes, 10; and pH was adjusted to 7·2 with HCl. When NMG⁺ was replaced with RbCl, NaCl, or CsCl, pH was adjusted with RbOH, NaOH, or CsOH, respectively. The standard bath solution contained (mM): NMG⁺, 140; Hepes, 10; MgCl₂, 1; CaCl₂, 1; and was adjusted to pH 7·4 with HCl. For recordings in the presence of different external Na⁺, K⁺, Rb⁺, or Cs⁺ concentrations, the NMG⁺ base external solution was used and the concentration of NMG⁺ was reduced as the cation concentration was elevated to maintain constant osmolarity. All chemicals were from Sigma.

Electrophysiological procedures

Coverslips containing cells were removed from the incubator before experiments and placed in a superfusion chamber (volume 250 µl) containing the control bath solution at 22-23°C. Current recording and data analysis were done using an Axopatch 200A amplifier and pCLAMP 6 software (Axon Instruments). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments). We have previously reported mean capacity transient decay rates between 46 and 62 µs (Fedida et al. 1996; Fedida, 1997). During the present study we report time constants of off-gating current decay (_off) of 0·4 ms and slower, so that recording bandwidth does not appear to be a limiting factor in the measurements. Capacity compensation and leak subtraction were routinely used, but series resistance (R_s) compensation was only rarely used. Measured series resistance was between 1 and 4 M for all recordings. When this changed during the course of an experiment, data were discarded. No difference between results with and without R_s compensation was observed. Data were sampled at 100-330 kHz and filtered at 5-10 kHz. All charge measurements (Q_off) were obtained by integrating the off-gating currents for 25 ms (except Fig. 1, 18 ms). Where current decay was slowed, full charge return may not have occurred during this time period. In those cases we measured a relative charge immobilization. As on-gating currents were unchanged, full return of charge occurred during the interpulse interval. Membrane potentials have been corrected for small junctional potentials that arose between pipette and bath solutions. For the solutions that contained NMG⁺ and 5 mM other monovalent cation (K⁺, Rb⁺, Na⁺, or Cs⁺) this amounted to -6·2 ± 0·5 mV for Cs⁺ and -6·1 ± 0·4 mV for Rb⁺ (means ± S.E.M., n = 6 trials) against solutions with 130 mM of these cations. For other solutions with 130 mM small cations versus NMG⁺ alone, measured junction potentials were 10 mV.

	RESULTS

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Measurement of off-gating currents in the WT and W472F channels

Gating currents from the mutated NCM channel overexpressed in HEK 293 cells using intracellular and extracellular NMG⁺ to prevent ionic flux were very similar to those observed with the wild-type (WT) hKv1.5 channel (Fig. 1A and B). On-gating currents appeared during depolarizations positive to -70 mV and increased in amplitude with larger depolarizations, then began to decay more rapidly at positive potentials (Fig. 1A). When repolarized to -100 mV, off-gating currents represent the return of gating elements as channels deactivate. In Fig. 1A and B these are shown as the downward current deflections after 12 ms duration depolarizations to between -60 and +80 mV. For small depolarizations to < -10 mV, off-gating currents reached a peak very rapidly and decayed rapidly and monoexponentially. After depolarizations to more positive potentials, the peak off-gating current was reduced and the time constant of relaxation of off-gating current (_off) slowed dramatically (Fig. 1A and B). A clear threshold for off-gating current slowing occurred at -10 mV, where the pore would normally be open for the channel to conduct ions (Fedida et al. 1993).

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Figure 1. Voltage dependent on- and off-gating currents from non-conducting mutant (NCM) W472F and wild-type (WT) hKv1.5 channels expressed in HEK cells in NMG+-containing solutions, and in the presence of 135 mM Cs+i-2·5 mM Cs On-gating currents (Ig,on) were recorded during depolarizations from -60 to +80 mV in 20 mV steps, and Ig,off on return to -100 mV. In A and C, families of gating currents are from NCM channels, and data in B and D are from WT hKv1.5 channels. An ionic Cs+ current can be recorded during depolarization from wild-type hKv1.5 channels (D). E, on- (Qon) and off-gating charge (Qoff) time courses obtained by integration of Ig,on and Ig,off for a +60 mV depolarization in A, B and C. F, histogram of charge ratios under the different experimental conditions, Qoff/Qon at +60 mV (mean ± S.E.M., n is indicated above the histograms).

When on-gating currents are integrated, the waveforms represent the time- and voltage-dependent movement of gating charge as channels progress towards the open state. The time course of on-charge movement (Q_on) is shown in Fig. 1E. There was no apparent difference between Q_on in the NCM and WT channels. When off-gating currents (I_g,off) are integrated, the slow return of charge is clearly seen in Fig. 1E where the rise of Q_off is delayed compared with Q_on (traces A and B). This slowing of Ig,off return from more positive potentials than the threshold for channel opening represents the slowed deactivation that occurs when permeant cations are absent from bath solutions (Chen et al. 1997). Recovery from this process was sufficiently slow that not all charge had returned during the period of integration. This gave a relative charge immobilization that we used in our experiments.

The slowing of off-gating charge return was largely prevented in both the NCM (Fig. 1C) and WT channels (Fig. 1D) when a 'physiological' Cs⁺ gradient of 135 mM Cs⁺_i-5 mM Cs was maintained across the cell membrane. In the WT channels, outward Cs⁺ current could be observed on depolarization (Fig. 1D), but on repolarization at E_Cs (Cs⁺ equilibrium potential), off-gating currents were fast and decayed rapidly, as we have previously shown (Chen et al. 1997). Due to the lack of any ionic Cs⁺ current in the NCM channel data, the time course of on- and off-charge movement could be directly compared (Fig. 1E, trace C), and the ratio of charge (Q_off/Q_on) calculated (Fig. 1F). There was still some slowing of off-gating currents occurring around the threshold for channel opening in the NCM channel, and this gave a rising phase to the off-gating currents recorded at more positive potentials. We have observed this phenomenon before (Chen et al. 1997) and it was absent in the WT channels, so may reflect the extreme rapidity with which NCM channels can inactivate (Yang et al. 1997). The effect of Cs⁺_i-Cs was to allow virtually complete charge return during the integration period, and thus Q_off/Q_on was 1·0 (Fig. 1F).

Species- and voltage-dependent modulation of Q_off by intracellular cations

In an attempt to localize the action of cations to one side or other of the ion channel pore we took advantage of the non-conducting nature of the W472F mutant and changed ion concentrations and species both intracellularly and extracellularly. Data in Fig. 2 were obtained using the external NMG⁺ solution and intracellular cation-containing solutions. Representative data for intracellular Rb⁺ and Na⁺ are shown in Fig. 2A and B. On-gating currents were very similar with the different cations. In the presence of 130 mM intracellular Rb⁺ (or K⁺), small outward currents were observed with large depolarizations. This represented ion flow through the endogenous delayed rectifier channels of HEK cells which usually amounted to about 200 pA of K⁺ current at +40 mV with a physiological ion gradient (Bouchard & Fedida, 1995). In the situation described in Fig. 2 the outward Rb⁺ driving force is very large with no external Rb⁺, and so we expected to observe small outward ion currents. The endogenous current did not significantly affect off-gating currents but prevented accurate measurement of Q_on for ratios in Fig. 2C. We observed that the endogenous ionic current was abolished by 1-2 mM extracellular TEA as described previously (Smith et al. 1996), and so Rb⁺ data in Fig. 2C were therefore obtained in the presence of 1 mM extracellular TEA, to which hKv1.5 channels are insensitive.

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Figure 2. Effect of intracellular cation species at 130 mM on NCM channel gating currents A and B, gating currents induced by depolarizations from -100 mV to between -60 and +80 mV in 20 mV steps, with internal Rb+ (A) or Na+ (B). NMG+ was the external cation. C, mean ratios of Qoff/Qon as a function of the voltage of depolarizing pulses. , NMG+; , Na+; , K+; , Rb+; , Cs+. D, off of Ig,off on repolarization. All data points in C and D are means ± S.E.M.; n as indicated in C.

Off-gating currents were differentially modulated by 130 mM of the various intracellular cations as illustrated by original data in Fig. 2A and B and mean data in Fig. 2C and D. Cs⁺ and Rb⁺ were most effective in preventing the slowing of Ig,off and facilitating the return of Q_off (Fig. 2C and D). Negative to the threshold for channel opening (-20 mV), the ratio of charge moved to that returned (Q_off/Q_on) was always close to 1·0 and the rate of decay of Ig,off was very rapid (_off = 0·5 ms). At opening potentials in the presence of Cs⁺ Q_off/Q_on ratios were preserved at around 1·0 (Fig. 2C) and with both Cs⁺ and Rb⁺ the time constants of decay remained rapid (Fig. 2D). At 130 mM, intracellular K⁺ and Na⁺ were less able to prevent the onset of charge immobilization and the voltage-dependent slowing of Ig,off that was most evident with intracellular NMG⁺.

Species- and voltage-dependent modulation of Q_off by extracellular cations

When the intracellular perfusion solution contained only NMG⁺ and different cations were included in the extracellular bath solution, only partial effects on reversing the immobilization of charge were observed. Original data obtained from gating current-voltage relations are shown in Fig. 3A-D. It can be seen that even with 130 mM Cs or Rb, quite marked off-gating current slowing was observed after depolarizations to more positive potentials. These data may be compared with corresponding intracellular cation data in Figs 2A and 4D. At 130 mM, Cs was about 50 % as effective as Cs⁺_i in preventing charge immobilization and slowing of Ig,off. Instead of a charge immobilization of 40 % with NMG⁺ only, 20 % Q_off immobilization was observed with 130 mM Cs (Fig. 3E). Similarly, _off was still quite slow, with values of about 4-6 ms (Fig. 3F). Rb was almost as effective as Cs, but again less effective than Rb⁺_i (Fig. 2D). Other ions were less effective than Cs⁺ in preventing charge immobilization and Ig,off slowing. Na at 70 mM had little effect above that of NMG⁺, and in this case data for 70 mM Cs are provided for comparison. These species effects of cations suggested that 130 mM intracellular cations were somewhat more effective at preventing the onset of immobilization than extracellular cations. The concentration dependence of these intracellular and extracellular effects are shown in Fig. 4 for Cs⁺.

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Figure 3. Effect of extracellular cation species on NCM channel gating currents A-D, families of gating currents obtained with different concentrations of extracellular cations as indicated. In each case the balance of extracellular monovalent cation was made up with NMG+, and 140 mM NMG+ was present internally. E, mean ratios of Qoff/Qon as a function of the voltage of depolarizing pulses. , 70 mM Na+; , 70 mM Cs+; , 130 mM Cs+; , 130 mM Rb+. F, off of Ig,off on repolarization; symbols as in C. All data points in C and D are means ± S.E.M.; n as indicated in C.

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Figure 4. Concentration-dependent modulation of Ig,off by Cs+ A-D, NCM channel gating currents during depolarizations to between -60 and +80 mV in 20 mV steps with different concentrations of Cs+i as indicated. 140 mM NMG+ was used in the external solutions. E-G, concentration-response curves for Ig,off modulation by Cs+i () or Cs (). In each experiment, Cs+ was present in either the extracellular or intracellular solution, but not both. E, peak Ig,off/peak Ig,on; F, Qoff/Qon; G, off of Ig,off after prepulses to +60 mV; in each case versus Cs+ concentration. Data were means from 3-7 cells as indicated adjacent to symbols in E. Lines were fitted to data after baseline subtraction, using an equation of the form: 1/(1 + (Kd/[Cs+])nH), where Kd represents the ion concentration which produced 50 % of the maximum response. In E for Ig,off/Ig,on, Kd = 42·5 ± 5·5 mM and nH = 1·2 ± 0·2 for Cs+i. The line fitted for Cs data was obtained by using the parameters for off from G. In F for Qoff/Qon, Kd = 3·1 ± 1·0 mM and nH = 1·4 ± 0·5 for Cs+i, and Kd = 131 ± 18 mM and nH = 1·5 ± 0·3 for Cs. In G for off, Kd = 20·2 ± 4·9 mM and nH = 0·63 ± 0·1 for Cs+i, and Kd = 358 ± 62 mM and nH = 0·54 ± 0·1 for Cs.

Concentration-dependent actions of Cs⁺_i and Cs

The dose-dependent actions of Cs⁺ included in the intracellular or extracellular media are shown in Fig. 4. Original data are shown for different concentrations of intracellular Cs⁺ in Fig. 4A-D. It can be seen that changes in the concentration of intracellular cation had little effect on the amplitudes or time course of Ig,on. However, the amplitude and time course of decay of the Ig,off was strongly affected by the concentration of Cs⁺_i. At 130 mM Cs⁺_i, off-gating currents at -100 mV almost mirrored the rapid rise and decay time course of Ig,on. The most important dose-dependent action of intracellular Cs⁺ was an alteration in the rate of charge return rather than effects on the total amount of charge returned during the recording period. We have made three measurements from these data in an attempt to quantify the action of Cs⁺_i () and Cs () on charge return. The ratio of peak Ig,off/Ig,on provided a sensitive measure of peak rate of charge return (Fig. 4E) along with the decay time constant of Ig,off (_off, Fig. 4G). A less sensitive measure was the relative immobilization of charge return, Q_off/Q_on (Fig. 4F). From the data in A-D and cumulated data in Fig. 4E -G it can be seen that both the intracellular and extracellular actions of Cs⁺ were of relatively low affinity. As the concentration of Cs⁺_i was increased from 5-130 mM, the peak Ig,off increased and the rate of decay accelerated. From fits of a Hill equation to the data (continuous lines in Fig. 4E -G) a K_d for Cs⁺_i of 42 mM was obtained for Ig,off/Ig,on and 20 mM for _off. For Q_off/Q_on (Fig. 4F), K_d values were lower (3·1 and 131 mM for Cs⁺_i and Cs, respectively), which simply reflected the fact that charge immobilization was not a sensitive measure of cation actions over such a short time scale. For all measured parameters, extracellular Cs⁺ was less effective than Cs⁺_i (Fig. 4E -G, open symbols). A K_d for Cs of 368 mM was obtained for _off, and the same value was used to give the dotted fit line for Ig,off/Ig,on in Fig. 4E.

	DISCUSSION

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In our earlier study we clearly demonstrated that cations strongly regulated the return of gating charge on repolarization (Chen et al. 1997). These experiments were carried out on conducting channels where it can be difficult to isolate the extracellular actions of cation species and concentration due to the effects of ions resident within the pore, or reaching the inner mouth from the outside - or vice versa. It seemed more definitive to examine the sidedness of ion regulation of charge return after preventing ion conduction altogether. We have attempted to do this in the present study by measuring off-gating current slowing and off-gating charge immobilization in the W472F non-conducting mutant to prevent ion conduction and allow us to alter ion concentration and species independently each side of the membrane. NCM channels have in general been thought to exhibit very similar voltage-dependent behaviour to WT channels, with the advantage that ion conduction can be prevented (Perozo et al. 1993). The hKv1.5 NCM channel behaved in a similar fashion to the WT channel as shown in Fig. 1, but was not quite identical. Cations could not prevent Q_off slowing in the NCM channels as effectively as in the WT channels, where _off of the off-gating current remained very fast (Chen et al. 1997). However, the NCM channel still showed strong regulation of Q_off by cations and did not appear to allow visible conduction of Cs⁺, Rb⁺, K⁺ (Yang et al. 1997), or outward Na⁺ currents (Starkus et al. 1997b). When NMG⁺_i-135 mM Na solution was used we did observe inward Na⁺ tail currents on repolarization, as has been reported recently for C-type inactivated channels and the W434F Shaker mutant (Starkus et al. 1997a, b). However, these had rather different waveforms to the Shaker mutant and only resulted in Q_off/Q_on ratios > 1·0 when Na was 130 mM. At lower concentrations of Na (70 mM, Fig. 3) we did not observe inward Na⁺ tail currents, nor were outward Na⁺ currents with Na⁺_i ever observed. These findings support the idea expressed below that, based on differences in channel structure, the hKv1.5 NCM channel may not behave identically to the Shaker NCM channel.

Modulation of charge return by cation species and concentration

The observation that we have made is that both intracellular and extracellular cations can impede the onset of charge immobilization and slowing. Intracellular cations were most effective at preventing this effect as indexed by the amount of charge returned, and the prevention of Ig,off slowing (Figs 2 and 4). When NMG⁺ alone was present 40 % charge immobilization occurred and _off was slowed 6-fold. We assume that the lack of effectiveness of NMG⁺ reflected the inability of the ion to access the pore at all, due to its large size. As the crystal radius of the accessible cations increased, more slowing was prevented in the sequence Cs⁺ > Rb⁺ > K⁺ > Na⁺ > NMG⁺. K⁺ was difficult to study due to permeation through a small endogenous delayed rectifier channel present in the cells (e.g. Rb⁺ Ig,on data in Fig. 2A), although this could be effectively blocked by low concentrations of TEA. Extracellular cations were less effective than intracellular cations with the sequence Cs⁺ > Rb⁺ > Na⁺ = NMG⁺. In this situation, Na was little more effective than NMG⁺. During the course of the study, Cs⁺ became the preferred ion and so we completed a full concentration-response curve for both Cs⁺_i and Cs (Fig. 4). Not only was Cs⁺ very good at preventing slowing and charge immobilization, but also its permeability was so low that no endogenous current was observed. The K_d for _off modulation was 20 mM for Cs⁺_i and 358 mM for Cs . From this we conclude that intracellular cations are significantly more effective at preventing charge slowing and immobilization in hKv1.5 than extracellular cations. Following on from this we suppose that binding sites exist on both the extracellular and intracellular sides of the channel with respect to the selectivity filter. At this narrowest point in the conduction pore the W472F mutation is likely to cause failure of cation permeation. At these putative intra- and extracellular binding sites, cations may modulate channel closing, or any processes such as residency in a long-lived closed state (Gomez-Lagunas, 1997; Jäger et al. 1998) that may secondarily slow channel closing. Both of the intracellular and extracellular sites are of relatively low affinity (mM), although we show here that the site with intracellular access has significantly higher affinity than the extracellular site. These data are quite consistent with current models of the K⁺ channel pore which propose cation binding sites at either end of the selectivity filter and a third site located deep within the inner vestibule (Durell & Guy, 1996; Doyle et al. 1998; Kiss et al. 1998).

Do the changes in off-gating current reflect the involvement of inactivation?

We have previously postulated that much of the slowing of off-gating charge return on repolarization, in the absence of cations, was caused either by an allosteric modulation of K⁺ channel deactivation, or an accelerated process of inactivation (Chen et al. 1997; Yellen, 1997). The present experiments have further defined the cation dependence of this modulation and the affinity of extracellular and intracellular sites for these effects. When the results are compared with ionic data presented in the accompanying paper (Fedida et al. 1999), some noticeable similarities are seen. The prevention of inactivation in WT channels by cations was size related, with the largest cation, Cs⁺, being most effective at preventing inactivation, and Na⁺ being the least effective. Intracellular cations were also more effective at preventing inactivation (K_d = 37 mM) than extracellular cations. The findings are closely paralleled by the sidedness, affinity and species effects of cations on gating charge return described here. In addition it was possible to define a K_d for the extracellular Cs⁺ modulation of charge return as 358 mM. These data are also consistent with our observation (Fedida et al. 1996) and those of others (Castle et al. 1994) that 4-aminopyridine binding at the inner mouth of the K⁺ channel pore can impede inactivation, perhaps in a similar manner to the cations.

While we believe the data are suggestive, they do not provide a compelling causal link. There are a number of reasons for this. The first is that the NCM channel is a mutated channel and cannot be compared directly with the WT channel. While the NCM channel probably gates in a very similar manner to WT channels, it is ion permeation that fails, probably due to disruption of a cation binding site deep in the pore. This is the very property that we seek to study in the NCM and WT Kv1.5 channels, so our comparison must be treated with caution. There are other important problems with an interpretation that the slowed charge return is due, in part or entirely, to accelerated inactivation. It has recently been reported that the non-conducting Shaker mutant W434F is permanently C-type inactivated, and that this may be the reason for its lack of conduction of ions (Yang et al. 1997). This creates a number of paradoxes, one of which has already been considered by Yang et al. (1997). They ask how a time-dependent shift in the voltage dependence of charge movement can reflect inactivation in W434F (Olcese et al. 1997), if the channel is permanently inactivated. Our data are not inconsistent with those of Yang et al. (1997) who propose that Shaker W434F is permanently inactivated at depolarized potentials. In the hKv1.5 NCM channel, charge immobilization is extremely rapid on depolarization to opening potentials when intra- and extracellular solutions contain only NMG, as suggested by data in Fig. 1A. We simply propose that in the absence of cations, formation of the inactivated state in the hKv1.5 NCM channel requires only a few milliseconds on depolarization. Unfortunately it is not possible to measure the onset of inactivation in WT channels when cations are omitted altogether, to directly compare WT and NCM channels.

This idea is supported by the comment that extracellular K⁺ or Rb⁺ increases current through heterotetramers of W434F (Yang et al. 1997), i.e. delaying onset of inactivation. However, although this can partially reconcile our NCM channel data with the Shaker W434F, our data also present a second paradox. If the charge immobilization in hKv1.5 does reflect inactivation, and this is largely prevented by cations (especially intracellularly), then presumably the NCM channels should become conducting when cations are included. Clearly they do not (Figs 2 and 3), so it may be that in hKv1.5, the rapid inactivation is not the only reason that the channels become non-conducting since it has already been suggested that inactivated channels can display altered permeabilities (Starkus et al. 1997). This is entirely reasonable, as the mutation is at a location very likely to disrupt a cation binding site within the pore (Durell & Guy, 1996). In the accompanying paper we have shown that a number of features of slow inactivation in hKv1.5 do not match classical C-type inactivation (Fedida et al. 1999). It is also possible that differences in channel structure between Shaker or Kv2.1, and hKv1.5 may underlie differences in the properties of the non-conducting mutants.

Conclusion

This work goes a long way towards defining the intracellular and extracellular regulatory roles of cations on gating charge movement in hKv1.5 channels. The NCM channel allowed a more complete analysis of the sidedness and species dependence of cation actions than conducting WT channels. We would like to stress that further experiments are still required to understand all the mechanisms by which cations modulate gating currents in this channel. Still, there are a number of similarities between the actions of cations on slow inactivation of ionic currents in WT channels and the actions of cations on gating charge return in hKv1.5 and the hKv1.5 NCM channel W472F. At the present time this leads us to favour the idea that at least part of the slowing of off-gating charge upon repolarization in the absence of intracellular or extracellular cations reflects a process of accelerated inactivation. We do not exclude other explanations, such as that there are multiple independent inactivation processes, of which the slow macroscopic type is only one. There may be additional closed states which the channel can occupy when cations are absent. Such states have recently been demonstrated for Shaker B (Gomez-Lagunas, 1997), Kv1.3 and Kv1.4 (Jäger et al. 1998). If cations modulate the occupancy of such states, the effects on off-gating charge that we observe could also be a direct action on deactivating K⁺ channels.

In the heart, Kv1.5 channels are important in cardiac repolarization, where modulation of its activity will regulate action potential duration and refractoriness. Our results demonstrate that gating of this channel, during deactivation, inactivation, or both, can be affected in subtle ways by local variations of cations at both the inner and outer mouths of the channel pore.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

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Acknowledgements

This work was supported by grants from the Heart and Stroke Foundations of Ontario, British Columbia and Yukon, and the Medical Research Council of Canada to D. F.

Corresponding author

D. Fedida: Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.

Email: fedida{at}interchange.ubc.ca

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