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¹ Department of Cellular and Physiological Sciences, University of British Columbia, 2146 Health Sciences Mall, Vancouver B.C. V6T 1Z3, Canada

	Abstract

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P/C-type inactivation of Kv channels is thought to involve conformational changes in the outer pore of the channel, culminating in a partial constriction of the selectivity filter. Recent studies have identified a number of phenotypic differences in the inactivation properties of different Kv channels, including different sensitivities to elevation of extracellular K⁺ concentration, and different state dependencies of inactivation. We have demonstrated that an alternatively spliced short form of Kv1.5, resulting in disruption of the T1 domain, exhibits a shift in the state dependence of inactivation in this channel, and in the current study we have examined this further to contrast the properties of inactivation from open versus closed states. In a TEA⁺-sensitive mutant of Kv1.5 (Kv1.5 R487T), 10 mM extracellular TEA⁺ inhibits inactivation in both full-length and T1-deleted channels, but does not inhibit closed-state inactivation in T1-deleted channel forms. Similarly, substitution of K⁺ and Na⁺ with Cs⁺ ions in the recording medium inhibits inactivation of both full-length and T1-deleted channel forms, but fails to inhibit closed-state inactivation of T1-deleted channels. Collectively, these data distinguish between open-state and closed-state inactivation, and suggest the presence of multiple possible mechanisms of inactivation coexisting in Kv1 channels.

(Received 28 March 2005; accepted after revision 14 July 2005; first published online 14 July 2005)
Corresponding author D. Fedida: 2146 Health Sciences Mall, Vancouver B.C. V6T 1Z3, Canada. Email: fedida{at}interchange.ubc.ca

	Introduction

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Recent studies have demonstrated that different Kv channel types can exhibit a broad spectrum of non-N-type inactivation phenotypes, with differences in sensitivity to extracellular K⁺ and TEA⁺, and differences in state dependence (Klemic et al. 1998, 2001; Jerng et al. 1999). Classical P/C-type inactivation of Shaker and its mammalian Kv1 homologues is generally not voltage dependent at potentials that saturate channel open probability, and is inhibited by extracellular TEA⁺ or elevation of extracellular K⁺ (Grissmer & Cahalan, 1989; Choi et al. 1991; Hoshi et al. 1991; Lopez-Barneo et al. 1993). In contrast, inactivation of Kv2.1 channels is accelerated by high concentrations of extracellular K⁺, and exhibits an apparent voltage dependence, such that inactivation is favoured at voltages near the V_1/2 of activation, and reduced at higher voltages (VanDongen et al. 1990; Klemic et al. 1998; Immke et al. 1999). This voltage dependence results in a characteristic upturned inactivation–voltage relationship that has led to the term ‘U-type inactivation’, and has been attributed to inactivation that occurs preferentially from partially activated closed states (Klemic et al. 1998, 2001; Kurata et al. 2001). Other channels, including the rapidly inactivating mammalian Kv4 family, also exhibit prominent closed-state inactivation that is accelerated by elevated extracellular K⁺ concentrations (Jerng et al. 1999; Bahring et al. 2001). There appears to be considerable diversity among non-N-type inactivation mechanisms of Kv channels (Rasmusson et al. 1998).

We have previously demonstrated that disruption of the T1 domain of the Shaker homologue Kv1.5 dramatically alters the inactivation phenotype of this channel (Kurata et al. 2001, 2002). Full-length Kv1.5 channels exhibit a voltage-independent inactivation mechanism, with properties similar to a classical mechanism of P/C-type inactivation (Fedida et al. 1999; Kurata et al. 2001). However, a truncated form of Kv1.5 (Kv1.5N209), that has been detected in murine and human cardiomyocytes (Tamkun et al. 1991; Attali et al. 1993; Fedida et al. 1993), exhibits a significant voltage dependence of inactivation that is very similar to that observed in Kv2.1 (Kurata et al. 2001, 2002). Kv1.5N209 also exhibits significant voltage dependence of recovery from inactivation, and rate-dependent cumulative inactivation properties that differ from the full-length channel, indicating that N-terminal truncation alters the state dependence of inactivation of Kv1.5 (Klemic et al. 1998; Kurata et al. 2001, 2002). Most importantly, N-terminal truncations of Kv1.5 remain the only known mutations to confer a U-shaped inactivation–voltage relationship in Kv channels. Therefore, from a biophysical perspective, comparisons of the inactivation properties of full-length Kv1.5 and truncated forms of the channel may provide novel insights into the properties of channel inactivation taking place at different stages in the activation pathway. Furthermore, despite the fact that truncated isoforms of Kv1.5 may arise by alternative splicing in cardiomyocytes (Attali et al. 1993), and reports of T1-deleted forms of Kv1.1 generated by post-translational proteolysis (Strang et al. 2001), there is little understanding of how the unique gating properties of truncated Kv1.5 channels may alter or contribute to the cardiac action potential.

In this study, we set out to compare the properties of inactivation of full-length Kv1.5 channels with the closed-state inactivation process that takes place in Kv1.5N209. To this end, we have characterized the modulation of inactivation and recovery from inactivation of full-length Kv1.5 and Kv1.5N209 channels using various experimental conditions that modulate inactivation in one or both channels. Our experiments identify several conditions that appear to selectively inhibit the inactivation mechanism present in full-length Kv1.5, and do not affect the closed-state inactivation process present in Kv1.5N209 channels. Collectively, the results suggest that inactivation of Kv1.5N209 channels is composed of two distinct processes: an inactivation mechanism similar to full-length Kv1.5, and a closed-state inactivation mechanism that is absent or minimal in full-length Kv1.5 channels and exhibits different regulation by permeant cations, extracellular TEA⁺, and voltage.

	Methods

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Cell culture

All cells were grown in Minimal Essential Medium (MEM) with 10% fetal bovine serum, at 37°C in an air–5% CO₂ incubator. Unless otherwise noted HEK-293 cell lines stably expressing full-length human Kv1.5 or the short form of human Kv1.5, Kv1.5N209, were used in all experiments. The Kv1.5N209 short form was generated by removal of the NcoI–NcoI fragment of Kv1.5 (Kurata et al. 2001). HEK-293 cells were stably transfected with full-length Kv1.5 or Kv1.5N209 cDNAs using LipofectACE reagent (Canadian Life Technologies, Bramalea, ON, USA).

Measurements of full-length and truncated Kv1.5 R487T mutant channels were carried out in transiently transfected HEK 293 cells. One day before transfection, cells were plated on glass coverslips in 35 mm Petri dishes with 20–30% confluence. On the day of transfection, cells were washed once with MEM with 10% fetal bovine serum. In order to identify the transfected cells efficiently, channel DNA was cotransfected with a vector encoding GFP. Channel DNA was mixed with pGFP (1: 1 ratio, 1 µg each) and 2 µl of LipofectAMINE 2000 (Canadian Life Technologies) in 100 µl of serum-free media, then added to the dishes containing HEK 293 cells. Cells were allowed to grow overnight before recording, and positive transfectants were identified at the time of recording using an epifluorescence attachment on the patch clamp recording apparatus.

Solutions

For recording in K⁺ conditions, patch pipettes contained (mM): NaCl, 5; KCl, 135; Na₂ATP, 4; GTP, 0.1; MgCl₂, 1; EGTA, 5; Hepes, 10; adjusted to pH 7.2 with KOH. The low extracellular K⁺ bath solution contained (mM): NaCl, 135; KCl, 5; Hepes, 10; sodium acetate, 2.8; MgCl₂, 1; CaCl₂, 1; adjusted to pH 7.4 with NaOH. The high extracellular K⁺ bath solution contained (mM): KCl, 135; Hepes, 10; dextrose, 5; MgCl₂, 1; CaCl₂, 1; adjusted to pH 7.4 with KOH. For recordings in Cs⁺ or Rb⁺ solutions, patch pipettes contained (mM): CsCl or RbCl, 135; MgCl₂, 1; EGTA, 10; Hepes, 5; adjusted to pH 7.2 with CsOH or RbOH. Bath solutions contained (mM): CsCl or RbCl, 135; Hepes, 10; dextrose, 10; MgCl₂, 1; CaCl₂, 1; and was adjusted to pH 7.4 with CsOH or RbOH. All chemicals were from Sigma Aldrich Chemical Co. (Mississauga, Ont., Canada).

Immunoblotting and antibodies

Canine heart tissue was kindly supplied by Dr David Van Wagoner of the Cleveland Clinic. All surgical procedures and experimental protocols were approved by the Cleveland Clinic Foundation Institutional Animal Care and Use Committee (Cleveland, OH, USA). Approximately 0.2 g of frozen heart was minced on dry ice and homogenized immediately in 5 ml lysis buffer (0.025 M phosphate buffer, pH 7.2 with, 10% glycerol and 0.5% IGEPAL (Sigma), 1 mM iodoacetamide, 0.2 trypsin inhibitor units ml^–1 aprotinin, 1 mM PMSF) using an Ultra-Turrax T25 homogenizer (IKA Laboratechnik, Germany). Nuclei and cell debris were pelleted at 1000 g for 10 min and the supernatant was run on SDS-PAGE gels for Western analysis. All homogenization and fractionations were carried out at 4°C. Blots were probed with a polyclonal anti-Kv1.5 antibody generated in our laboratory, a rabbit antibody against the C-terminus of hKv1.5 (aa 537–553 EQGTQSQGPGLDRGVQR; 1: 10 000). This antibody was chosen for its unique sequence region at the C-terminus that is not shared with other Kv channels. It shares 12 of 17 residues with canine Kv1.5 and detected canine Kv1.5 under a number of experimental conditions (Fedida et al. 2003). Detection was by HRP-labelled goat anti-rabbit antibody (Jackson Immuno Research, PA, USA) and Renaissance Western blot Chemiluminescence Reagent Plus (NEN).

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, and perfused with bathing solution throughout the experiments. Whole-cell current recording and data analysis were done using an Axopatch 200A amplifier and pCLAMP 8 software (Axon Instruments). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, FL, USA). Electrodes had resistances of 1–3 M when filled with control filling solution. Capacity compensation and 80% series resistance compensation were used in all whole-cell recordings. The mean capacitance and series resistance from 82 cells studied was 16.2 ± 0.6 pF and 2.4 ± 0.1 M (before series resistance compensation). No leak subtraction was used when recording currents, and zero current levels are denoted by the dashed lines in the current tracings. Data were sampled at 10–20 kHz and filtered at 5–10 kHz. Membrane potentials have not been corrected for small junctional potentials between bath and pipette solutions. Throughout the text data are shown as mean ± S.E.M.

Data analysis

The voltage dependence of inactivation of Kv1.5 and Kv1.5N209 were fitted with Boltzmann functions of the form y = 1/(1 + exp[(V – V_1/2)/k]) + C, respectively, where V_1/2 represents the voltage at which 50% inactivation occurred, V is the membrane potential, and k is the slope factor that reflects the steepness of the voltage dependence. C represents the fraction of non-inactivating channels at potentials where inactivation was most complete. Slopes of the upturn of inactivation–voltage relationships were determined by linear regression, and are listed in the text with 95% confidence intervals for the slope. Throughout the text, data are presented as mean ± S.E.M., and tests for significance between two groups were carried out using Student's t test or the Mann-Whitney U test.

	Results

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N-terminal deletions of Kv1.5 allow additional pathways of inactivation

We have previously reported that deletion of residues in the N-terminus of Kv1.5, particularly within the T1 domain, can dramatically alter the inactivation properties of the channel (Kurata et al. 2001, 2002). RNA of splice variants encoding an N-terminally truncated form of Kv1.5 (Kv1.5N209) have been isolated from cardiac tissue of several species, suggesting that Kv1.5N209 may be present in vivo at certain developmental stages, although protein expression of truncated channel isoforms has not yet been demonstrated (Tamkun et al. 1991; Attali et al. 1993; Fedida et al. 1993). In recent studies we have used an antibody (Fig. 1A) directed against the C-terminus of Kv1.5 to establish the presence of Kv1.5 in canine and mouse heart (Fedida et al. 2003; Brunet et al. 2004) Following on from this work, we have also detected multiple immunoreactive bands, suggestive of multiple forms of Kv1.5 in lysates of canine atrial tissue from several different animals (Fig. 1B). The predicted size of full-length canine Kv1.5 is 71 kDa, although in Western blots we find that it normally runs at a slightly higher molecular weight of 83 kDa, suggesting an additional 12 kDa arising from glycosylation. The predicted size of a truncated form corresponding to Kv1.5N209 is 44 kDa, and similar post-translational processing of Kv1.5N209 and full-length Kv1.5 could account for the 58 kDa band observed consistently in Western blots (Fig. 1B).

View larger version (37K): [in this window] [in a new window]   Figure 1.  Multiple isoforms of Kv1.5 in canine cardiac tissue A, C-terminal epitope recognized by the antibody used for Kv1.5 detection. B, Western blot of lysates of canine atrial tissue from six separate animals. A Lowry assay was run in triplicate for each sample and 15 µg per sample were loaded in each lane. The C-terminal Kv1.5 antibody detects species at 83 and 58 kDa.

View larger version (24K): [in this window] [in a new window]   Figure 2.  Multiple pathways of inactivation in Kv1.5 A, inactivation–voltage relationships were determined by whole-cell patch clamp recordings from HEK 293 cells stably expressing Kv1.5 or Kv1.5N209. From a holding potential of –80 mV, cells were depolarized briefly to +60 mV (P1), followed by a 2 s rest at –80 mV. Cells were then depolarized for 5 s between –70 and +70 mV in 10 mV steps (P2), followed by a 500 ms test pulse to +60 mV (P3). B, traces to +60 mV (left) or 0 mV (right) in cells expressing full-length Kv1.5 or Kv1.5N209 have been normalized to their respective peak currents, to directly compare the time course of inactivation. C, as an index of inactivation during P2, peak current during the test pulse (P3) was normalized to peak current during the control pulse (P1).

Despite these recent advances in our understanding of state-dependent inactivation in various Kv channels, it remains unclear whether closed-state inactivation is a fundamentally different process from P/C-type inactivation, a process generally considered to be strongly coupled to channel opening. To describe some experimental data, models in which closed- and open-inactivated states can interconvert appear to be adequate (Scheme 1; Kurata et al. 2001). However, other experimental work has implied that a distinct mechanism of closed-state inactivation (U-type) can coexist with P/C-type inactivation in Shaker (Scheme II; Klemic et al. 2001). In this study, we present a number of experiments to investigate the biophysical properties of the closed-state inactivation mechanism revealed by deletion of the Kv1.5 N-terminus, and to determine whether this process is equivalent to the P/C-type inactivation mechanism observed in full-length Kv1.5 channels.

(Scheme 1)

(Scheme 2)

Enhanced closed-state inactivation in Kv1.5N209

Careful examination of the inactivation properties of full-length Kv1.5 and Kv1.5N209 at voltages insufficient to significantly activate channels also emphasizes the altered state-dependence described for Kv1.5N209. From our prior studies we have established the activation relationships for full-length Kv1.5 and Kv1.5N209 at 10 and 5 mV intervals (Kurata et al. 2001, 2002), such that the activation V_1/2 values of Kv1.5 and Kv1.5N209 are –10.8 ± 0.8 and –20.3 ± 1.7 mV, respectively (Kurata et al. 2002; Fig. 1). Cells stably expressing either full-length Kv1.5 or Kv1.5N209 were held at voltages between –50 and –20 mV, and pulsed briefly (15 ms) to +60 mV at 2 s intervals to examine the onset of inactivation at these intermediate voltages (Fig. 3A and C). At all voltages above –50 mV, Kv1.5N209 exhibited significantly more inactivation than full-length Kv1.5 (Fig. 3B and D). Importantly, the activation V_1/2 of Kv1.5N209 is shifted by –10 mV relative to full-length Kv1.5 (Kurata et al. 2001). The mean activation of full-length Kv1.5 was only significant at –20 mV (0.094 ± 0.016) and not significant at more negative potentials, whereas the mean activation of Kv1.5N209 was significant at –30 mV (0.078 ± 0.06) and –20 mV (0.54 ± 0.10), with discernible activation only at –35 mV (0.03). However, even when this difference is taken into account, considerably more inactivation was observed in Kv1.5N209. For instance, a membrane voltage of –30 mV elicited very little inactivation (18 ± 2%) over 40 s in full-length Kv1.5 channels, and only –20 mV, a potential which activated significant numbers of channels, resulted in obvious inactivation, suggesting that this might be open-state dependent (Fig. 3C). In contrast, pulses from –40 mV in Kv1.5N209, a potential at which no significant activation occurred, resulted in 47 ± 5% inactivation of Kv1.5N209 (Fig. 3D), which is significantly greater than that found at –30 mV in full-length Kv1.5 (P < 0.05, Fig. 3). These data illustrate the importance of open-state inactivation in full-length Kv1.5 and closed-state inactivation in Kv1.5N209 channels.

View larger version (28K): [in this window] [in a new window]   Figure 3.  Closed-state inactivation of full-length Kv1.5 and Kv1.5N209 Cells stably expressing either full-length Kv1.5 or Kv1.5N209 were held at voltages between –50 and –20 mV, and were pulsed for 15 ms to +60 mV at 2 s intervals to monitor the extent of channel inactivation (5 mM K+i/135 mM K+o ionic conditions). Sample data in A illustrate inactivation of full-length Kv1.5 with a holding potential of –30 mV, and C illustrates inactivation of Kv1.5N209 with a holding potential of –40 mV. Mean data at multiple voltages for full-length Kv1.5 and Kv1.5N209 are illustrated in B and D, respectively.

To demonstrate a P/C-type mechanism of inactivation in Kv1.5, and to compare the regulation of inactivation by extracellular cations in Kv1.5 and Kv1.5N209, we have examined the effects of elevation of extracellular K⁺ on inactivation in both channels (Fig. 4). This experiment was prompted by reports that other K⁺ channels with a U-shaped inactivation–voltage relationship, including Kv2.1 and Kv3.1, often exhibit more pronounced inactivation in the presence of high extracellular K⁺ (Klemic et al. 1998, 2001), in contrast with previous studies of Shaker and other channels exhibiting a P/C-type inactivation mechanism (Lopez-Barneo et al. 1993; Molina et al. 1997). Inactivation was measured in both full-length Kv1.5 and Kv1.5N209, in two different extracellular K⁺ concentrations (5 and 135 mM), at two different conditioning voltages (+10 and +60 mV in full-length Kv1.5; –20 and +60 mV in Kv1.5N209). The ‘intermediate voltages’ (+10 and –20 mV) used in Fig. 4 were selected based on the position of the ‘foot’ of the inactivation–voltage relationships for full-length Kv1.5 and Kv1.5N209 (Fig. 2C). To quantify inactivation, cells were given a 100 ms control pulse to 60 mV (P1), rested for 2 s at –80 mV, stepped to one of the two conditioning voltages for 5 s (P2), followed by a test pulse to +60 mV (P3). Sample data are shown normalized to the P1 test pulse, to correct for differences in current magnitude due to driving force changes in different extracellular K⁺ conditions. Therefore, the magnitude of current in the P3 test pulse reflects the extent of inactivation during the conditioning pulse (P2). Because the effects of extracellular K⁺ in Kv1.5 are quite small, experiments were conducted with a paired design, with summarized data presented in the bar graphs in Fig. 4C and F. The bar graphs show the mean difference in fractional inactivation observed in 135 mM K⁺ and 5 mM K⁺ in full-length Kv1.5 (filled bars) and Kv1.5N209 (open bars). In these graphs a positive number indicates less inactivation in 135 mM versus 5 mM K⁺.

View larger version (20K): [in this window] [in a new window]   Figure 4.  Effects of extracellular K+ on inactivation kinetics in Kv1.5 and Kv1.5N209 Inactivation during 5 s pulses was quantified as described in Fig. 2. HEK 293 cells stably expressing (A and D) Kv1.5 or (B and E) Kv1.5N209 were pulsed to a P2 voltage of either 60 mV (lower panels) or a voltage at the foot of the inactivation–voltage relationship (upper panels, +10 mV for Kv1.5 and –20 mV for Kv1.5N209, determined from the inactivation–voltage relationships in Fig. 2C). The extent of inactivation was determined by the ratio of the peak currents in P3 and P1. C and F, mean data of differences in the P3/P1 ratio in 5 mm K+o versus 135 mm K+o in both Kv1.5 and Kv1.5N209.

Modulation of Kv1.5N209 inactivation properties by permeant ions and external TEA⁺

Because the effects of K⁺ on Kv1.5 inactivation are quite small, we have examined the effects of other permeant or blocking ions known to inhibit P/C-type inactivation in Shaker and its mammalian homologues. With this approach, we have also identified several experimental manipulations that appear to selectively inhibit ‘open-state’ inactivation, without interfering with the inactivation process present in truncated forms of Kv1.5, and these are discussed in the sections that follow. To introduce our reasoning in these experiments, it is useful to consider in advance the expected effects of inhibition of open state versus closed state inactivation on the properties of the inactivation–voltage relationship. Experimental conditions that inhibit P/C-type inactivation from the open state generally do so with very weak voltage dependence. An example is the inhibition of P/C-type inactivation by extracellular TEA⁺ (see below), and the net result is expected to be an upward shift of the plateau of the inactivation–voltage relationship. In Kv1.5N209, we have suggested that macroscopic inactivation results from a combination of inactivation from the open state and closed states. In this case, manipulations that inhibit closed-state inactivation would have strong effects on channel inactivation at intermediate voltages (where closed-state inactivation is maximal), but weaker effects at high voltages (where closed-state inactivation contributes minimally to the overall time course of inactivation). Therefore, the overall result of conditions that inhibit closed-state inactivation in Kv1.5N209 would be a reduction in the slope of the ‘upturn’ of the inactivation–voltage relationship (i.e. a reduction in the magnitude of the ‘upturn’ relative to the peak non-inactivated current).

In full-length Kv1.5 channels, Rb⁺ substitution for K⁺ mildly inhibits inactivation during the 5 s pulses used in our experiments, resulting in an upward shift of the plateau of the inactivation–voltage relationship by roughly 10% (Fig. 5A and C). In Kv1.5N209 channels, Rb⁺ also inhibits inactivation (Fig. 5B); however, the slope of the upturn of the inactivation–voltage relationship is clearly blunted relative to the data collected with K⁺ as the permeating ion (Fig. 5D). In Rb⁺ recording conditions, the slope of the upturn of the inactivation–voltage relationship was described with a linear fit with a slope of 1.0 ± 0.1% per 10 mV, significantly smaller than the slope in 5/135 mM K⁺ or 135/135 mM K⁺ conditions (P < 0.05). This result suggests that Rb⁺ is able to inhibit inactivation from both open and closed states in Kv1.5N209.

View larger version (23K): [in this window] [in a new window]   Figure 5.  Rb+ substitution attenuates both open-state and closed-state dependent inactivation A and B, HEK 293 cells expressing either full-length Kv1.5 or Kv1.5N209 were pulsed from a holding potential of –80 mV to +60 mV for 6 s, to observe the time course of inactivation. C and D, inactivation–voltage relationships for Kv1.5 and Kv1.5N209 were determined as described in Fig. 2; however, K+ was replaced with Rb+ in both the pipette and extracellular solutions. In D, the inactivation–voltage relationships for full-length Kv1.5 have been superimposed in grey, for comparison.

View larger version (16K): [in this window] [in a new window]   Figure 6.  Extracellular TEA+ does not affect closed-state inactivation A, cells expressing Kv1.5N209 were pulsed from a holding potential of –80 mV to +60 mV, to observe the time course of inactivation in control conditions, or in the presence of 10 mM extracellular TEA+. B, inactivation–voltage relationships for Kv1.5N209 R487T were determined as described in Fig. 2, in the presence or absence of 10 mM extracellular TEA+.

View larger version (24K): [in this window] [in a new window]   Figure 7.  Cs+ substitution selectively attenuates open-state dependent inactivation A and B, cells stably expressing full-length Kv1.5 or Kv1.5N209 were pulsed from a holding potential of –80 mV to +60 mV with either K+ or Cs+ as the permeant ion. Inactivation–voltage relationships for Kv1.5 (C) and Kv1.5N209 (D) were determined as described in Fig. 1, with K+ or Cs+ in both the pipette and extracellular solutions. In D the inactivation–voltage relationships collected for full-length Kv1.5 are superimposed in grey, for comparison.

Modulation of recovery from inactivation by prepulse potential and extracellular K⁺

As described above, our experiments were initiated with the working hypothesis that truncated Kv1.5 channels inactivate by multiple mechanisms, with P/C-type inactivation favoured at depolarized potentials where channel open probability is maximal. In contrast, at intermediate voltages near the V_1/2 for channel activation, channels show greater occupancy of partially activated closed states than at higher voltages, and closed-state inactivation becomes favourable. We hypothesized that if channels access distinct inactivated states at different voltages (i.e. via closed-state versus open-state inactivation), then the recovery kinetics may depend on the voltage at which channels are inactivated. Similar findings have been reported in an earlier study of recovery from inactivation in Shaker, in which inactivation of channels at strongly depolarized voltages resulted in different recovery kinetics than inactivation at voltages closer to the V_1/2 of channel activation (Klemic et al. 2001). Recovery from inactivation was measured using a modification of the protocol shown in Fig. 2A. From a holding potential of –80 mV, cells were depolarized to +60 mV (P1, 20 ms), briefly repolarized to –80 mV, depolarized for 7 s to a variable potential to inactivate the channels (P2, 7 s), repolarized to a recovery voltage of –110 mV for a variable duration, and finally depolarized briefly to +60 mV (P3, 20 ms). The ratio of P3/P1 current magnitudes provides an index for the extent of recovery at various time intervals.

We examined the time course of recovery in full-length Kv1.5, after inactivating the channel at either +60 or +10 mV. Summary data are plotted in Fig. 8C, together with parameters of bi-exponential fits to the time course of recovery. In full-length Kv1.5, the rate of recovery from inactivation is relatively independent of the inactivating voltage, as are the relative contributions of the fast and slow components of the recovery time course. In Kv1.5N209 channels, the time course of recovery was examined after inactivating prepulses to either +60 or –20 mV (chosen because at –20 mV the extent of closed-state inactivation is maximal). Inactivating prepulses of –20 mV result in different kinetics of recovery than when channels are inactivated at +60 mV (Fig. 8D). In particular, inactivation at –20 mV increases the amplitude of the fast component of recovery, and decreases the amplitude of the slow component of recovery when compared with the recovery kinetics observed following depolarizations to +60 mV. The net effect is the observation of a slightly more rapid rate of recovery when Kv1.5N209 channels are inactivated at –20 mV versus +60 mV. This result suggests that at –20 mV, channels preferentially inactivate via a different mechanism (which recovers faster) than at +60 mV (which recovers more slowly).

View larger version (25K): [in this window] [in a new window]   Figure 8.  Effects of prepulse potential on recovery from inactivation A and B, after a brief control pulse to +60 mV (P1), HEK 293 cells stably expressing full-length Kv1.5 or Kv1.5N209 were depolarized to +60 mV or an intermediate voltage (+10 mV in full-length Kv1.5, –20 mV in Kv1.5N209) for 7 s (P2), repolarized to –110 mV for a variable duration, followed by a brief test pulse (P3) to +60 mV. C and D, recovery from inactivation during the recovery period was parameterized with the ratio of P3/P1, and records from individual cells were fitted with double exponential equations. The fit parameters are included on the plots.

View larger version (32K): [in this window] [in a new window]   Figure 9.  Effects of prepulse potential and extracellular K+ on recovery from inactivation Recovery from inactivation was measured under various extracellular K+ conditions, and with various prepulse voltages in cells expressing full-length Kv1.5 (A, B) or Kv1.5N209 (C, D), with the same protocol described in Fig. 8. The time course of recovery was fitted with a bi-exponential equation. Fit parameters shown as insets in each panel represent the relative weights of the fast and slow recovery components. Detailed fit parameters are presented in Table 1.

View this table: [in this window] [in a new window]   Table 1. Effects of extracellular K+ on recovery from inactivation in Kv1.5 and Kv1.5N209

	Discussion

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Slow inactivation of mammalian Kv1 channels is thought to be voltage independent and coupled to channel opening, and this mechanism is generally referred to as C-type or P/C-type inactivation (Hoshi et al. 1991; Olcese et al. 1997; Klemic et al. 2001; Kehl et al. 2002). The voltage independence of this process is reflected in a flat inactivation–voltage relationship at voltages that saturate channel open probability (Hoshi et al. 1991), as shown in Fig. 2C by the open symbols. The characterization of short forms of Kv1.5 (Kurata et al. 2001) and Kv1.5 T1-deletion mutants (Kurata et al. 2002), together with recent studies in Shaker (Klemic et al. 2001), suggest that Shaker family K⁺ channels are able to exhibit at least two distinct inactivation phenotypes. As suggested by Klemic et al. (2001), we have referred to these here as P/C-type and U-type inactivation. We have previously demonstrated that disruption of the T1 domain of Kv1.5 channels dramatically alters their inactivation properties, imparting a U-shaped inactivation–voltage relationship similar to Kv2.1 channels (Kurata et al. 2001, 2002). To date, N-terminal truncations and T1 domain mutations of Kv1.5 remain the only ones identified that impart a U-shaped inactivation–voltage relationship to a Kv channel, and may provide unique insights into the mechanism(s) underlying closed-state inactivation of Kv channels (Kurata et al. 2002), as a direct comparison of the two inactivation mechanisms is permitted in a single channel. In particular, while Kv1.5N209 inactivates significantly more than full-length Kv1.5 channels at intermediate voltages, the extent of inactivation closely approaches the full-length channel at high voltages (e.g. +60 mV or greater, Fig. 2C). For this reason, we have been able to suggest that N-terminal truncation of Kv1.5 leaves the P/C-type inactivation mechanism of the full-length channel largely unaltered, but adds additional pathways for channel inactivation that contribute significantly to the overall inactivation time course at intermediate depolarizations (Kurata et al. 2001). The question that we have tried to address here, using this unique property of Kv1.5, is whether this additional pathway of U-type inactivation is a variant of the P/C-type mechanism, or whether its properties are sufficiently discrete to require different structural mechanisms.

Separation of P/C- and U-type inactivation mechanisms

Functionally, these two inactivation phenotypes differ greatly. P/C-type inactivation occurs predominantly from the open state, is inhibited by extracellular K⁺ or TEA⁺, and exhibits relatively slow and weakly voltage-dependent recovery from inactivation (Hoshi et al. 1991; Rasmusson et al. 1995, 1998; Klemic et al. 2001). Channels exhibiting U-type inactivation properties, however, appear to exhibit preferential inactivation from partially activated closed states, rapid and strongly voltage-dependent recovery from inactivation, and in some channel types accelerated inactivation with elevation of extracellular K⁺ or TEA⁺ (Klemic et al. 1998, 2001). Our experiments have extended these results to other cations with somewhat surprising results. Both Rb⁺ and Cs⁺ slow Kv1.5 inactivation during sustained depolarizations at positive potentials, when compared with K⁺ as the permeating ion (Figs 5A and 7A; (Fedida et al. 1999), and this mechanism appears to be preserved in Kv1.5N209 (Figs 5B and 7B). The inactivation mechanism in full-length Kv1.5 accounts for the Cs⁺-sensitive component of inactivation in Kv1.5N209. Shortening of the N-terminus allows inactivation via an additional Cs⁺-insensitive pathway, accounting for the deeper inactivation and indistinguishable upturn of the inactivation–voltage relationship in Kv1.5N209 whether K⁺ or Cs⁺ is the permeant ion (Fig. 7D). Thus, our results provide some confirmation/validation of our earlier interpretation of Kv1.5N209 gating, where we suggested that the excessive inactivation observed at intermediate depolarizations was the result of accelerated inactivation from intermediate closed states in the activation pathway (Kurata et al. 2001).

We have previously suggested that Cs⁺ traps Kv1.5 channels in the P-type inactivated state and prevents the structural transition of channels into deeply inactivated P/C-type states (Wang & Fedida, 2001). Interestingly, our data suggest that the inactivation process from closed states underlying the Kv1.5N209 phenotype is inaccessible to Cs⁺ modulation. The importance of this experiment is that closed-state inactivation in Kv1.5N209 is demonstrated to be distinct from the P/C-type inactivation process, as suggested in Shaker (Klemic et al. 2001). Rb⁺, on the other hand, appears to diminish all components of Kv1.5N209 inactivation (Fig. 5), and this may reflect its known action on channel open probability (Demo & Yellen, 1992). Prolonged occupancy of Rb⁺ in the selectivity filter will inhibit/delay the conformational changes underlying P/C-type inactivation. Furthermore, by increasing the stability of the open state, the closed-state inactivation responsible for the upturn of the inactivation–voltage relationship will be indirectly diminished, as Rb⁺-occupied channels will dwell less often in closed states.

As with Cs⁺, addition of extracellular TEA⁺ was able to significantly inhibit P/C-type inactivation in both full-length Kv1.5 R487T and Kv1.5 R487T/N209, but the extent of the upturn of the inactivation–voltage relationship remained essentially unaltered under these experimental conditions (Fig. 6). A simple interpretation of these observations is that Cs⁺ and extracellular TEA⁺ ions selectively inhibit the P/C-type inactivation mechanism observed in full-length Kv1.5 channels, and do not affect the closed-state inactivation mechanism revealed by truncation of the Kv1.5 N-terminus. Since it is well understood that extracellular TEA⁺ has specific actions at the outer pore of the potassium channel that compete with the ability of the channel to adopt the locally constricted state recognized as P/C-type inactivation (Yellen et al. 1991; Ikeda & Korn, 1995; Molina et al. 1997; Loots & Isacoff, 2000) the results suggest that the ‘U-type’ closed-state inactivation process does not involve the same conformational changes of the channel as those responsible for P/C-type inactivation. Unfortunately, the conformational changes associated with ‘U-type’ or closed-state inactivation remain poorly understood. Structure–function studies in Kv1.5 have shown that N-terminal intracellular domains, and particularly the highly conserved T1 domain, are somehow involved in allowing the channel to access these closed-inactivated states (Kurata et al. 2002). Other studies in Kv2.1 and its auxiliary -subunits have suggested an important role for proline residues in the inner cavity in regulating state-dependent inactivation (Kerschensteiner et al. 2003). Clearly, further experiments are required to understand the role of intracellular domains, and the structural basis for differences in regulation by extracellular cations, in the closed-state inactivation mechanisms of Kv channels.

Recovery from inactivation

Although less direct, the effects of prepulse voltage and extracellular K⁺ concentration on the biexpoenential recovery kinetics of Kv1.5 and Kv1.5N209 provide further evidence for the presence of multiple distinct pathways of inactivation. In general, multiple experimental manipulations that enhanced closed-state inactivation of Kv1.5N209 increased the amplitude of the fast component of recovery from inactivation in this channel. Closed-stated inactivation was maximized by conditioning channels at an intermediate voltage (–20 mV, Fig. 8), which optimizes channel occupancy in partially activated closed states. Increasing extracellular K⁺ also promoted inactivation of Kv1.5N209, and this presumably arose from an effect on a ‘U-type’ or closed-state inactivation process specific to Kv1.5N209, since extracellular K⁺ exerts an opposite effect on inactivation of the full-length Kv1.5 channel. Consistent with this, elevation of extracellular K⁺ appears to promote the closed-state inactivation processes in Kv2.1 and Kv3.1 (Klemic et al. 1998, 2001). Both of these experimental manipulations (high extracellular K⁺ and conditioning at intermediate voltages) enhanced the amplitude of the fast component of recovery in Kv1.5N209, suggesting that the closed-state inactivation process maximized in these conditions recovers with rapid kinetics. Since the kinetics of recovery (particularly the balance of fast and slow components) appear to be influenced by the conditions during the inactivating pulse, the different inactivation processes leading to multiple components of recovery are probably mutually exclusive rather than interconvertable.

A significant difficulty in interpreting the kinetics of recovery from inactivation is that there is also a bi-exponential time course of recovery in full-length Kv1.5 channels (Figs 8 and 9, and Table 1). While we are unsure of the fundamental mechanisms governing the time course of recovery, bi-exponential recovery kinetics have been frequently reported in other Kv channels including Shaker (Levy & Deutsch, 1996; Klemic et al. 2001). The study of Klemic et al. (2001) interpreted the multi-exponential recovery kinetics as a reflection of distinct recovery kinetics from two different mechanisms of inactivation (U-type and P/C-type). We have adopted similar reasoning in our interpretation of recovery kinetics in full-length Kv1.5 and Kv1.5N209. That is, there is clearly a rapid component to recovery from inactivation in these channels, and we have interpreted changes in the amplitude of this component to reflect changes in the extent of an alternative (closed-state) mechanism of inactivation in Kv1.5N209. Important, however, is the observation that full-length Kv1.5 channels clearly exhibit a rapid component to recovery from inactivation, but show little evidence of any significant closed-state inactivation (Fig. 3). They do not exhibit a U-shaped inactivation–voltage relationship (Fig. 2), or excessive cumulative inactivation during trains of repetitive depolarizations (Kurata et al. 2001). Thus, it is unclear whether the rapid component of recovery from inactivation can be tied to a distinct inactivation process in full-length Kv1.5, as has been suggested for Shaker (Klemic et al. 2001). The kinetics of recovery from inactivation of the full-length Kv1.5 channel do appear to be slightly altered by extracellular K⁺ or prepulse voltage (though not to the same extent as Kv1.5N209; Figs 8 and 9), and this could reflect contributions of multiple inactivation processes in full-length Kv1.5 (e.g. P/C-type and a minor contribution of U-type inactivation) that are differentially regulated by voltage and permeant ions. This raises the possibility that the significant effects of N-terminal deletions on Kv1.5 inactivation arise by specifically enhancing (or altering the state dependence of) an inactivation mechanism that is normally present in full-length Kv1.5 channels but overshadowed by P/C-type inactivation.

Conclusion

N-terminal truncation of Kv1.5 exerts significant effects on the inactivation properties of the channel that can be explained by acceleration of inactivation from partially activated closed states. We have demonstrated that the closed-state inactivation process revealed in N-terminally deleted forms of Kv1.5 is insensitive to cations (Cs⁺ and extracellular TEA⁺) that inhibit inactivation of the full-length channel. Our data also suggest that experimental conditions favouring closed-state inactivation mechanisms alter the kinetics of recovery from inactivation. The differential regulation of closed-state inactivation by cations and voltage suggests that multiple distinct mechanisms of inactivation can co-exist in truncated forms of Kv1.5.

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	Acknowledgements

 
This work was supported by grants from the Heart and Stroke Foundations of British Columbia and Yukon, and the CIHR to D.F. H.K. was supported by predoctoral fellowships from the CIHR and Michael Smith Foundation for Health Research. We thank Anu Khurana for assistance with cell culture and David Van Wagoner for the supply of canine atrial tissue.

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