Correspondence to: David Fedida, Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3 Canada. Fax:(604) 822-6048 E-mail:fedida{at}interchange.ubc.ca.
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Abstract |
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Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5N209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V1/2 of -19.5 ± 0.9 mV for activation and -35.7 ± 0.7 mV for inactivation, compared with a V1/2 of -11.2 ± 0.3 mV for activation and -0.9 ± 1.6 mV for inactivation in full-length Kv.15. Kv1.5
N209 channels exhibited several features rarely observed in voltage-gated K+ channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and "excessive cumulative inactivation," in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5
N209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5
N209 showed 3035% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:17791789) in which an absence of the NH2 terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K+ current diversity in human heart and many other tissues.
Key Words: potassium channel, Kv1.5, gating, inactivation, NH2-terminal deletion
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INTRODUCTION |
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The Kv1.5 (KCNA5) channel is a mammalian homologue of Shaker that is widely expressed throughout the cardiovascular system and elsewhere. It is found in human adult and fetal hearts (
What is less generally appreciated is that Kv1.5 channels, like KvLQT1 (
The region of the NH2 terminus that is absent in the short form of the channel is important for a number of fundamental properties. The first 200 amino acids mediate the interaction of the channel with ß subunits (100 amino acids in length. The T1 domain has been shown to prevent heteromultimerization of different Kv channel subfamilies (
Despite their long NH2-terminal sequence, Kv1.5 channels have no NH2-terminal ball structure, and are thought to inactivate by slower mechanisms that which we group together here under the term "C-type mechanism" (
In this study, we have characterized the gating properties of the short NH2-terminal form of human Kv1.5. Our study shows that the NH2 terminus of Kv1.5 exerts effects on both channel activation and inactivation. Of particular interest was that the lack of the NH2 terminus resulted in a U-shaped voltage dependence of C-type inactivation. We show that this is related to a shift in the state dependence of channel inactivation toward inactivation from closed states, and we believe that this is the first characterization of modulation of C-type inactivation by the NH2 terminus of a Shaker family K+ channel.
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MATERIALS AND METHODS |
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Cell Preparation and Transient Transfection
All cells were grown in MEM with 10% FBS, 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, hKv1.5N209, were used in all experiments. The Kv1.5
N209 short form was generated by removal of the NcoI-NcoI fragment of Kv1.5 (see Fig 1 A). HEK 293 cells were stably transfected with full-length hKv1.5 or Kv1.5
N209 cDNAs using LipofectACE reagent (Canadian Life Technologies).
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In some experiments, a Mouse ltk- (L) cell line was used as a transient expression system due to its lack of endogenous voltage-gated K+ channel subunits. 1 d before transfection, cells were plated on glass coverslips in 35-mm petri dishes with 2030% confluence. On the day of transfection, cells were washed once with MEM with 10% FBS. To identify the transfected cells efficiently, channel DNA was cotransfected with the vector pHook-1 (Invitrogen). This plasmid encodes the production of an antibody to the hapten phOX which, when expressed, is displayed on the cell surface. Channel DNA was mixed with pHook-1 (1:1 ratio, 1 µg each) and 2 µl of LipofectAMINE 2000 (Canadian Life Technologies) in 100 µl of serum-free media, and then added to the dishes containing mouse L cells. Cells were allowed to grow overnight before recording. 1 h before experiments, cells were treated with beads coated with phOX. After 15 min, excess beads were washed off with cell culture medium, and cells that had beads stuck to them were used for electrophysiological recordings.
Solutions
Patch pipettes contained the following (in mM): 5 NaCl, 135 KCl, 4 Na2ATP, 0.1 GTP, 1 MgCl2, 5 EGTA, and 10 HEPES (adjusted to pH 7.2 with KOH). The bath solution contained the following (in mM): 135 NaCl, 5 KCl, 10 HEPES, 2.8 sodium acetate, 1 MgCl2, and 1 CaCl2, (adjusted to pH 7.4 with NaOH). All chemicals were from Sigma-Aldrich.
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 2223°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). Electrodes had resistances of 13 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 dotted lines in the current tracings. Data were sampled at 1020 kHz and filtered at 510 kHz. Membrane potentials have not been corrected for small junctional potentials between bath and pipette solutions. Throughout the text, data are shown as mean ± SEM, and tests for significance between two groups were performed using t test.
Data Analysis and Modeling
The voltage dependence of activation and inactivation of Kv1.5 and Kv1.5N209 were fit with Boltzmann functions of the form: y = 1/(1 + exp[(V1/2 - V)/k]) and y = 1/(1 + exp[(V - V1/2)/k]) + C, respectively, where V1/2 represents the voltage at which 50% activation or 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 noninactivating channels at potentials where inactivation was most complete. The model was constructed using ScoP and ScoPfit (version 3.51; Simulation Resources), and based on the allosteric model of inactivation proposed by
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RESULTS |
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Amino Acids 1209 Modulate the Activation Properties of Kv1.5
The loss of the first 209 amino acid residues in the short form of Kv1.5 corresponds to elimination of the NH2 terminus nearly to the S1 transmembrane domain, and includes deletion into the T1B domain (Fig 1 A). The T1A, T1B, and T1C regions correspond to residues 121150, 156225, and 233260 respectively, based on alignments with Shaker (N209), as well as the expression of T1-deleted channels in Xenopus oocytes (
N209 appeared very similar to those seen in the full-length Kv1.5 channels (Fig 1B and Fig C), although single exponential fits to the activation time course in Kv1.5
N209 reveal slightly faster time constants of activation than in the full-length channel (Fig 1 E). However, the activation curve of Kv1.5
N209 channels does exhibit a leftward shift relative to the full-length channel. The half-activation voltage (V1/2) was shifted from -11.2 ± 0.3 mV in full-length Kv1.5 channels (n = 5) to -20.9 ± 1.6 mV in Kv1.5
N209 (n = 5, Fig 1 D).
Characterization of the Inactivation-voltage Relationship of Kv1.5N209
The most striking differences between the full-length Kv1.5 and the shorter Kv1.5N209 channels are in their respective inactivation properties (Fig 2). Inactivation-voltage relationships were measured isochronally with a standard double pulse protocol, consisting of a 5-s conditioning pulse to various potentials to inactivate the channel, followed by a brief test pulse to +60 mV, where the conductance-voltage relationship is saturated. Thus, the measured amplitude of the test pulse current is proportional to the number of available (noninactivated) channels. Each double pulse protocol was preceded by a brief 100-ms pulse to +60 mV, which was not long enough to cause further inactivation, but ensured that changes in pipette access resistance and channel rundown during the lengthy protocol did not significantly affect the measured inactivation-voltage relationships. The half-inactivation voltage was shifted from -19.5 ± 0.9 mV in full-length channels to -35.7 ± 0.7 mV in Kv1.5
N209. Furthermore, lack of residues 1209 imparts a dramatic U-shaped voltage dependence to the inactivation-voltage relationship (Fig 2 C). The inactivating pulses used in these experiments as illustrated in Fig 2 (A and B) were limited to 5 s, as longer pulses made the completion of the entire protocol impractical. Since inactivation remains incomplete even after depolarizations up to 20 s in duration (Fig 2 D), the inactivation-voltage relationships presented do not represent a steady state. However, as shown in Fig 2 D, the observed U-shaped voltage dependence of inactivation is preserved even after 20 s inactivating pulses. Two voltage pulses to +60 and 0 mV were given to full-length Kv1.5 channels (Fig 2 D, top) and Kv1.5
N209 channels (Fig 2 D, bottom). These were followed by a test pulse to +60 mV to measure the residual current. In the Kv1.5
N209 channels, the test pulse current after the 0 mV prepulse falls below that after the +60 mV prepulse. This indicates a U-shaped inactivation-voltage relation in the short form of Kv1.5, which was not observed in full-length channels.
Over the time courses examined, Kv1.5N209 channels appear to inactivate more completely than full-length Kv1.5 channels (Fig 2C and Fig D). There is an increase in the maximum level of inactivation during 5-s depolarizations, from a mean of 49 ± 2% to 79 ± 2% in Kv1.5
N209. In a previous study of full-length Kv1.5, we have reported that 10-s depolarizations gave a mean reduction of 60% at +60 mV (
N209 short form can inactivate more completely than the full-length channel, and the half-inactivation voltage of the channel is modified, as well as the overall shape of the inactivation-voltage relationship. These findings suggest alternative or additional pathways for inactivation in the short form of the channel.
As noted, the T1 domains have been implicated in prevention of interfamily subunit heteromultimerization (300 pA at +60 mV so that for the size of currents illustrated in Fig 1 and Fig 2, 90% of the observed current can be accounted for by Kv1.5
N209 channel subunits. To confirm that the currents elicited in HEK 293 cells arose from homotetramers of the Kv1.5
N209 subunit, as opposed to heterotetramers containing HEK 293 cell endogenous channel subunits, we transiently expressed Kv1.5
N209 in Mouse L cells, which showed little or no endogenous voltage-gated K+ current. The measured inactivation-voltage relationship of Kv1.5
N209 exhibited the same features observed in the HEK 293 cell expression system: an upturn of the relationship, and a leftward shift of the half-inactivation voltage. The maximum percent inactivation attained during 5-s pulses is 75.3 ± 3% at -10 mV compared with 46 ± 5% inactivation at +60 mV, the latter value is almost identical with that reached in full-length Kv1.5. The half-inactivation potential is -18.5 ± 1.1 mV in full-length Kv1.5, and shifted to -30.2 ± 0.9 mV in Kv1.5
N209. These data support the conclusion that the unique inactivation behavior of Kv1.5
N209 is an intrinsic property of the short form of the channel, and is not cell-type dependent or due to heteromultimerization with endogenous channel subunits.
Closed-state Inactivation and Excessive Cumulative Inactivation
The only previous report of a U-shaped voltage dependence of inactivation in Kv channels is for Kv2.1 and its heteromers with Kv5.1 and Kv9.3 (N209, and Kv2.1, normalized to the peak current at each voltage. The currents evoked in Kv2.1 and Kv1.5
N209 are qualitatively very similar, each displaying a highly evident voltage dependence to the observed amount of inactivation. In agreement with the inactivation-voltage relations (Fig 2 C) there is a faster rate and greater relative amount of inactivation at 0 mV than at more positive potentials. Full-length Kv1.5 channels do not exhibit such a voltage dependence, and mean data are summarized in Table 1. Here, inactivation of full-length Kv1.5 and Kv1.5
N209 during 20-s depolarizations have been fit to a biexponential decay function with fast (
1) and slow (
2) time constants. The difference between inactivation rates in the two channels is explained at 0 mV by the almost twofold faster rate of
2 in Kv1.5
N209 than in Kv1.5.
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A second consequence of inactivation from late closed states is the observation of excessive cumulative inactivation, where more complete inactivation is observed when channels are repeatedly shuttled between positive and negative potentials. This allows channels to repeatedly pass through closed states, and results in more complete inactivation than would be observed if channels were held continuously in the open state. We have tested for excessive cumulative inactivation in full-length Kv1.5 and Kv1.5N209, to ascertain whether closed state inactivation in the
N209 short form may explain the U-shaped inactivation-voltage relation. Representative records are shown in Fig 4, in which cells were repetitively pulsed to a test potential (+60 or 0 mV) for 90 ms followed by a 10-ms repolarization period at -80 mV. The peak currents during each test pulse were compiled and their decay (shown as the open symbols) compared with the current decay observed during a continuous pulse to the test potential (solid line). Clearly, in Kv1.5
N209, rapid shuttling with a 90:10 duty ratio between 60 and -80 mV (Fig 4 B) results in excessive cumulative inactivation, in that the peak current measured during the repetitive pulses falls below the current level during the continuous pulse at +60 mV. It is of note that shuttling the potential between 0 and -80 mV does not lead to excessive inactivation (Fig 4 D), which is consistent with this potential being close to the minimum of the inactivation-voltage relationship (Fig 2 C). Full-length Kv1.5, in contrast, exhibits no excessive cumulative inactivation under either condition.
To further characterize excessive cumulative inactivation in this channel, cells expressing full-length Kv1.5 or Kv1.5N209 were subjected to repetitive depolarizations (-80 mV to 0 or +60 mV) in which the interpulse interval was held constant at 20 ms, but the pulse duration and number of pulses were varied such that the total depolarization time was 5 s (Fig 5 A). The residual current observed after each pulse train was normalized to the current remaining at the end of a continuous 5-s pulse. In full-length channels, data denoted by the closed circles indicates that the amount of inactivation during a train of repetitive depolarizations from -80 to +60 mV never significantly exceeds the amount of inactivation that occurs during a continuous pulse (i.e., normalized current doesn't fall below 1.0). This observation is consistent with the assertion that the amount of inactivation observed is related to the total time spent depolarized, implying that in full-length Kv1.5, inactivation occurs primarily from the open state. Also, when pulsed repetitively between -80 and 0 mV, the relationship was shifted to longer pulse durations (open circles) than for the pulse trains between -80 and +60 mV. This observation is most likely due to the slower activation kinetics at 0 mV relative to +60 mV (Fig 1), and again supports the idea that closed-state inactivation does not contribute substantially to full-length Kv1.5 inactivation, since channels would be expected to remain significantly longer in closed states when pulsed to 0 mV than when pulsed to +60 mV.
When the same protocol was applied to Kv1.5N209, pulse trains to +60 mV with intermediate pulse durations (10200 ms) resulted in substantially more inactivation than a continuous 5-s pulse. This finding suggests that the amount of inactivation observed is related to the number of times a channel shuttles between open and closed states. In simple terms, a high pulse frequency passes channels through closed states more often, and results in a greater amount of inactivation. This experiment suggests a marked shift in the state dependence of inactivation in the short form of Kv1.5, as inactivation in Kv1.5
N209 appears to occur from closed states, with or without a component of inactivation from the open state. An examination of individual data tracings during the 80-ms depolarizations in Kv1.5
N209 shows that, after the first pulse, currents rise extremely rapidly in all subsequent pulses (Fig 5 B). The instantaneous rising phase of the second pulse current in Fig 5 B is a driving force effect on the fraction of channels that have failed to close, however, there is also a reduction in the delay preceding channel opening, which indicates that channels lack the time to completely deactivate during the interpulse interval, and only reach early closed states in the deactivation pathway. Kv1.5 and Kv1.5
N209 channels deactivate with similar kinetics, and with deactivation time constants of roughly 6 ms at -80 mV one would expect slightly more than half of the channels to close during a 10-ms repolarizing pulse, which corresponds to the magnitude of the instantaneous current component relative to the total current activated (Fig 5 B). If channels are inactivating from closed states, this observation suggests that these closed states are late in the activation pathway, very near to the open state.
Recovery from Inactivation
We also observed that the voltage dependence of recovery from inactivation is markedly altered in Kv1.5N209 channels (Fig 6). HEK 293 cells expressing full-length Kv1.5 or Kv1.5
N209 were subjected to 7-s inactivating pulses at +60 mV, and the recovery at holding potentials between -50 and -110 mV was observed for up to 30 s. The difference in the voltage dependence of recovery is immediately evident upon examination of the representative traces presented in Fig 6 A. In Kv1.5
N209, recovery is nearly complete within 2 s at -110 mV, but is extremely slow with a holding potential of -50 mV. In full-length Kv1.5, the time course of recovery appears to vary far less over the same voltage range. Summary data are shown for a range of recovery potentials between -50 and -110 mV for full-length Kv1.5 in Fig 6 B, and Kv1.5
N209 in Fig 6 C. The holding potential has a fairly minor effect on recovery from inactivation in full-length Kv1.5. However, in Kv1.5
N209, recovery from inactivation exhibits a much stronger voltage dependence. Overall, the half-time for current recovery in Kv1.5
N209 was found to vary roughly 20-fold over the voltage range examined, whereas recovery of full-length Kv1.5 was found to vary only approximately threefold. This difference was found to be significant at potentials of -50, -65, -95, and -110 mV (Fig 6 D, P < 0.05).
Excessive Inactivation of Kv1.5N209 under Cardiac Action Potential Clamp
To compare cumulative inactivation in full-length Kv1.5 and Kv1.5N209 using a more physiological stimulus, we recorded an action potential from a human ventricular myocyte (at 1 Hz) and subjected transfected HEK 293 cells to this waveform repetitively at frequencies of either 1 or 1.67 Hz. The waveform is shown in the inset to Fig 7 A. In both channels, inactivation accumulates over the course of the pulse trains, seen as a progressive decrease in the peak current during trains of action potential clamps (Fig 7A and Fig B). Peak current during each pulse has been normalized to the current during the first pulse and plotted against the respective pulse number to illustrate the relative current decays over time at the two pulsing frequencies (Fig 7C and Fig D). At both pulse rates, peak currents decrease substantially more in Kv1.5
N209 than in full-length Kv1.5, which suggests that in the physiological setting of the human heart, inactivation is likely to accumulate to a greater degree in Kv1.5
N209 than in the long form of the channel.
Kinetic Models of Inactivation Models Predicting U-shaped Inactivation-voltage Relationships
Apart from the Ca2+ currentdependent inactivation of L-type Ca2+ channels, models that predict a U-shaped inactivation-voltage relationship can be divided into two classes. The first of these is characterized by voltage-dependent microscopic inactivation rates between the open state and one or more inactivated states (Scheme 1 and Scheme 2). An example is the three-state cyclic model of
The second class of model describing a U-shaped inactivation-voltage relationship is characterized by accelerated inactivation from an intermediate state in the activation pathway. Examples include the model of
We considered two specific models in this second class, as shown in Scheme 3 and Scheme 4. In Scheme 3, the channel is proposed to occupy multiple open states in a voltage-dependent manner, with inactivation occurring most readily from the first open state; i.e., k3, k4, and k5 are voltage-independent transitions, but k4 is > k5. If depolarization favors occupancy of the second open state, the model predicts a U-shaped voltage dependence of inactivation, and excessive cumulative inactivation, because strongly depolarized channels will repeatedly pass through the first open state during trains of repetitive depolarizations, giving them an increased likelihood of inactivating. However, the model does not predict a dissociation of the voltage dependencies of activation from inactivation as we observed in Kv1.5N209.
In Scheme 4 (Fig 8), the model structure parallels that of Scheme 3, but the model contains a single open state. Voltage-dependent microscopic activation and deactivation rates predict greater occupancy of a late closed-state at intermediate potentials. If k3 > k2, the model predicts more rapid inactivation at intermediate potentials. This model also predicts excessive cumulative inactivation, as channels will repeatedly pass through the late closed state during repetitive depolarizations. In the context of Scheme 4, we would hypothesize that deletion of the NH2 terminus of Kv1.5 causes accelerated inactivation from late closed states, similar to the model proposed for inactivation of Kv2.1. The model of N209. As well, it is in agreement with a single open state for Shaker channels (
Building a Kinetic Model of Inactivation in Kv1.5 and Kv1.5N209
With the above considerations in mind, we constructed a model based upon the previously published model of Kv2.1 inactivation. The model consists of 12 states: five closed states, five closed-inactivated states, one open state, and one inactivated state (Fig 8). Horizontal transitions between states are governed by exponential functions of voltage, and the model assumes four independent transitions along the activation pathway. Vertical transitions were assumed to be independent of voltage. Forward (ko) and backward (kob) rates between the open state and final closed state were governed by exponential functions of voltage, and were determined by fitting the voltage dependence of time constants of single exponential fits of deactivation and activation rates (data not shown). The resulting activation transitions reproduce the experimentally observed kinetics and voltage dependence of activation in the full-length channel (Fig 9 A), with simulations yielding a V1/2 of activation of -12 mV. Also, current records from long 20-s pulses to +60 mV in the full-length channel were used to estimate the forward and backward rates of inactivation from the open state (koi, kio), based on the assumption that most inactivation was occurring from the open state in the full-length channel. The parameter g is included to preserve microscopic reversibility. We then systematically varied the rates of inactivation from the open state and closed states, to investigate the potential influence of closed-state inactivation in the different forms of Kv.1.5.
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We found that the rate of inactivation from the open state was the prime determinant of the time course of inactivation at depolarized potentials. This is expected, due to the strong bias toward the open state with strong depolarizations predicted by our activation parameters (Fig 9 B). Our simulations predict that above 10 mV, >90% of channels occupy the open state (Fig 9 B). In the formulation of the model presented in Fig 8, simulations of the inactivation-voltage relations resulted in behavior qualitatively resembling the full-length channel, as long as the closed-state inactivation rate was roughly equal to or less than the rate of open-state inactivation. However, the model produces essentially monoexponential kinetics of inactivation, in contrast to the biexponential kinetics observed experimentally. The only noted alteration of the inactivation-voltage relationship upon deceleration of the closed-state inactivation rate is a depolarizing shift of the V1/2 of inactivation (Fig 9 D). To adequately describe the dissociation between the half-activation and half-inactivation potential (roughly an 8-mV hyperpolarizing shift) in full-length Kv1.5, we estimated a closed-state inactivation rate of 0.00008 ms-1 (kci; Fig 8). The inactivation-voltage relationship derived from this simulation (Fig 9 A) was fit with a Boltzmann function to yield a V1/2 of -20 mV, which is close to the value observed experimentally in the full-length channel.
Incorporating the U-shaped Voltage Dependence
A number of possible changes to the model can result in a U-shaped inactivation-voltage relationship. Initially, we examined the effects of destabilization of inactivation from the open state. Deceleration of the rate of open-state inactivation not only results in a U-shaped inactivation-voltage relationship (Fig 9 C, inset), but also results in very slow inactivation that poorly reproduces the time course of inactivation observed at depolarized potentials (Fig 9 C, solid lines). In addition, a far more significant acceleration of closed-state inactivation is required to approximate the time course of inactivation experimentally observed with strong depolarizations. For example, deceleration of inactivation from the open state by 10-fold (chosen because it is sufficient to generate a mildly U-shaped inactivation-voltage relationship) requires acceleration of closed-state inactivation by roughly 250-fold to reproduce the experimentally observed time course of inactivation at +60 mV (Fig 9 C, broken lines). The result of such an acceleration is more profound inactivation at intermediate potentialsin our example, we see complete inactivation within 2.5 s at 0 mV (Fig 9 C)and consequently an exaggerated U-shape of the inactivation-voltage relationship.
A second kinetic change that could account for a U-shaped inactivation-voltage relationship is simply acceleration of inactivation from closed states. This possibility was particularly attractive, thanks to unique insights gained through comparisons of the N209 channel with the full-length channel. As noted, inactivation of Kv1.5
N209 was more complete than for full-length Kv1.5 at intermediate potentials, but gradually approached the level observed in the full-length channel at more positive potentials. This can be explained by the presence or enhancement of an inactivation mechanism in addition to the primarily open-state inactivation observed in full-length Kv1.5. More importantly, deeper inactivation at intermediate voltages in Kv1.5
N209 is a rigid prediction of the model, and is not necessarily predicted by a model based on destabilization of inactivation from the open state.
Acceleration of inactivation transitions from closed-states of the channel results in a gradual leftward shift of the inactivation-voltage relationship, without affecting the activation relationship, and further acceleration results in a U-shaped steady-state inactivation relationship (Fig 9 D). Acceleration of closed-state inactivation to 0.0013 ms-1 results in a 17-mV dissociation between half-activation and half-inactivation potentials, and a marked U-shaped inactivation-voltage relationship (Fig 9 A; it is important to note that we have not considered the 10-mV hyperpolarizing activation shift in Kv1.5N209, and hence the V1/2 of the Boltzmann fit in Fig 9 D reproduces the enhanced dissociation of inactivation from activation, but not the true half-inactivation potential in Kv1.5
N209).
Excessive Cumulative Inactivation
The parameters derived using the approach described above also allowed effective simulation of excessive cumulative inactivation in full-length Kv1.5 and Kv1.5N209. Simulations of the repetitive 90-ms depolarizations (+60 mV) and 10-ms repolarizations (-80 mV) described in Fig 4 are shown in Fig 10 A. Clearly, the parameters derived for the full-length channel predict little or no excessive cumulative inactivation. In contrast, the parameters derived for Kv1.5
N209 predict excessive cumulative inactivation that is maintained throughout the 20-s pulse train with the absence of any crossover, as observed experimentally.
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The allosteric factor, f, reflects the extent to which inactivation is favored in partially activated states of the channel (Fig 8). It affects the half-inactivation voltage, since f governs the relative rates of inactivation and recovery at potentials where few or no channels reach the open state. For the same reason, f also exerts a very obvious effect on the voltage dependence of recovery from inactivation. Having roughly determined the rates of inactivation from late closed states, we used the rates of recovery at extremely negative potentials (i.e., -110 mV; Fig 6) to fix a rate for recovery from the earliest closed state. This line of reasoning was based on the assumption that extremely hyperpolarized recovery potentials would result in the vast majority of channels dwelling in their earliest closed state, and is corroborated by our observation of a single-exponential recovery time course at hyperpolarized recovery potentials. Finally, the allosteric factor f can be fixed by studying its effects on the predicted voltage dependence of recovery.
This approach can be used to adequately fit the voltage dependence of recovery in both full-length Kv1.5 and Kv1.5N209, however, it does not predict the crossover of their respective recovery rates at -80 mV, as was observed experimentally (Fig 6). To reproduce this feature of our experimental data, slight changes must be made to the modeled rates of recovery from inactivation (compare kic for the long and short forms of Kv1.5 in legend for Fig 8, and simulations shown in Fig 10B and Fig C). Finally, although this model predicts a strong voltage dependence of recovery in Kv1.5
N209 (Fig 10B and Fig C), it does not identically reproduce the voltage dependence of recovery observed experimentally (Fig 10 C and Fig 6B and Fig C). Most likely, this difficulty arises because of our simplification of the Kv1.5 activation pathway, as the model implies that the voltage-dependent occupancy of different closed-states governs the voltage dependence of the recovery pathway.
It is widely held that the steps involved in Kv1 channel activation are far more complex than those depicted here (
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DISCUSSION |
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A Change in the State Dependence of Inactivation in the Short Form of Human Kv1.5
We have shown that the activation, inactivation, and recovery properties of Kv1.5N209 are significantly modified from those of the long form, or full-length Kv1.5. Activation is affected only in a minor way, with a small, but significant, hyperpolarizing shift in the half-activation potential, and a decrease in the slope (Fig 1). The
N209 short form of Kv1.5 lacks almost the entire cytoplasmic NH2-terminal domain and a significant portion of the T1 intersubunit assembly domains, including all of T1A and most of the T1B portion (
T1 Shaker mutant (
The striking observation that we have made here is that the short form of Kv1.5 shows a marked shift in the state dependence of its inactivation from that found in the full-length channel. Our experimental data show that inactivation in full-length Kv1.5, as in most voltage-gated K+ channels, increases with voltage and eventually stabilizes, so that the inactivation-voltage relationship reaches a basal level at depolarized potentials. In Kv1.5, for very long pulses this is at 40% of the maximum current (
N209, inactivation shows a prominent upturn when studied in both HEK 293cells and mouse L cells (Fig 2), a property that has only been described before in WT Kv2.1 channels and its heteromultimers (
N209 demonstrate a voltage dependence to its inactivation equivalent to that seen in Kv2.1 (Fig 3 and Table 1). Kv1.5
N209 also shows excessive cumulative inactivation (Fig 4 and Fig 5) and significant voltage-dependent recovery from inactivation (Fig 6 C), although this is less marked than for Kv2.1 (
N209 at intermediate potentials with a nadir at around -20 or -10 mV, and a relief of inactivation at more positive potentials (Fig 2). Finally, these altered properties of Kv1.5
N209 result in a marked increased in accumulation of inactivation during repetitive depolarizations using a human cardiac action potential as the stimulus waveform (Fig 7).
Structural Basis for Closed State Inactivation in Kv1.5N209
Our data suggests that the NH2 terminus of Kv1.5 is involved in destabilizing the transition between the closed and closed-inactivated states of the channel. Several NH2-terminal deletions and point mutations have been described that modulate activation gating kinetics and the voltage dependence of activation in Shaker, but none have characterized direct modulation of the slow inactivation gating of a Shaker homologue (N209 occurs via classical C-type inactivation and, hence, can be modulated by pore occupancy. The current view of slow inactivation is that it involves conformational changes of the outer pore mouth, and recent studies suggest that interactions between residues in the S4 and S6 transmembrane segments are also critical in preventing closure of the slow inactivation gate until after channel opening (
Some preliminary studies in our laboratory have shown that the distal Kv1.5 NH2 terminus (roughly the first 120 residues) is not required for the maintenance of normal inactivation gating, suggesting that residues within T1 are involved in the modulation of inactivation. The implied coupling of the NH2 terminus to conformational changes in distal regions of the protein is interesting in light of recent reports postulating that the NH2-terminal T1 tetramer hangs away from the inner mouth of the channel pore of Shaker channels (
Model of Kv1.5N209 Inactivation
Our comparison of full-length Kv1.5 and Kv1.5N209 provided the important insight that the time course of inactivation is accelerated in Kv1.5
N209 at intermediate potentials, but slows and approaches that of full-length Kv1.5 at more extreme depolarizations. The simplest interpretation of this observation is that the
N209 deletion allows an additional avenue for inactivation, which is favored at intermediate potentials, and our kinetic model supports this hypothesis. In particular, we have shown that the inactivation-voltage relationship and excessive cumulative inactivation of Kv1.5
N209 can be reproduced by accelerating the rates of inactivation from closed-states of the channel, and allowing inactivation to proceed from the open state at rates observed in the full-length channel. The result of this model is that the truncated channel behaves similarly to full-length Kv1.5 at depolarized potentials, but inactivates more rapidly and completely (over the time intervals examined) at intermediate potentials where few channels are open. In addition, the acceleration of inactivation from closed-states predicts enhanced dissociation of inactivation from activation (Fig 9A and Fig D). All of these features of the model are consistent with experimental data.
The most fundamental insight gained from our modeling is that the NH2 terminus of Kv1.5 modulates the inactivation phenotype primarily by regulating the amount of inactivation that occurs from late closed-states. In addition, our model suggests that the mechanism for this acceleration is a shift in the allosteric coupling of inactivation to transitions through the activation pathway. Although the mechanism implied by our model is not the only possible way to accelerate closed-state inactivation, this mechanism is also consistent with our observation of enhanced voltage dependence of recovery in Kv1.5N209. Furthermore, the concept of allosteric coupling of inactivation and/or recovery to transitions in the activation pathway is implied in a number of recent models of inactivation of voltage-gated K+, Na+ and Ca2+ channels (
Although the model structure is essentially identical to that of N209 inactivation during repetitive depolarizations occurs from the open state of the channel, rather than late closed states. Inactivation from late closed states provides an additional inactivation mechanism that accounts for the deeper inactivation observed due to increased state occupancy at intermediate depolarizations or during repetitive pulse trains.
An important issue for consideration is why we observe relatively minor dissociation of activation from inactivation in Kv1.5N209 in spite of the proposed rapid rates of closed-state inactivation. This is especially striking in comparison with Kv4.1 in which the half-activation and half-inactivation potentials are dissociated by as much as 40 mV (
Physiological Implications of Closed State Inactivation
Of particular interest in the present study is that NH2-terminally truncated forms of Kv1.5 have been identified in murine and human cardiac myocytes that may arise from an unusual splicing mechanism within the Kv1.5 gene structure (
We have not studied the effects of NH2-terminal deletions of other Kv1 channels, although Kv1.1, 1.2, and 1.3 all possess alternative start sites homologous to M210 in Kv1.5. There have been a number of studies of NH2-terminal deletions of Shaker and its Kv1 homologues, and none have reported any significant disruption of inactivation, so we suspect that these effects may be unique to Kv1.5. Since our data suggests that deletion of the NH2 terminus affects the depth of inactivation, it seems likely that differential expression of this channel could alter the properties of outward K+ currents in tissues such as cardiac muscle and vascular smooth muscle. For example, the expression of Kv1.5N209 could increase the sensitivity of action potential duration to heart rate, as inactivation of Kv1.5
N209 is enhanced by repetitive depolarizations, and during action potential clamp experiments (Fig 7). Also, maximal inactivation of Kv1.5
N209 occurs at potentials near the plateau of the cardiac action potential, so small physiological or pathophysiological changes in action potential shape could significantly affect inactivation of the channel. In addition, the T1 domain is required to maintain the specificity of subfamily subunit assembly, and the Kv1.5
N209 isoform will lack binding sites for both ß subunits (
-actinin-2 (
-actinin-2 has been implicated in modulating the expression level of Kv1.5 (
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Acknowledgements |
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We thank Dr. Rolf Joho for his gift of Kv2.1, and Qin Wang for assistance in preparing the cells.
This study was supported by grants from the Heart and Stroke Foundations of British Columbia and Yukon, and the CIHR to D. Fedida. H. Kurata was supported by a University Graduate Fellowship from the University of British Columbia.
Submitted: 12 February 2001
Revised: 1 August 2001
Accepted: 2 August 2001
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References |
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