Correspondence to: Stephen W. Jones, Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106. Fax:(216) 368-3952 E-mail:swj{at}po.cwru.edu.
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Abstract |
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The 1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members (
1G and
1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat
1I stably expressed in HEK293 cells. We found several kinetic differences between
1G and
1I, including some properties that at first appear qualitatively different. Notably,
1I tail currents require two or even three exponentials, whereas
1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for
1I, even for relatively strong depolarizations. Despite these differences, gating of
1I can be described by the same kinetic scheme used for
1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 512-fold slower for
1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability.
Key Words: inactivation, activation, T-current, LVA channel, kinetic models
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INTRODUCTION |
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T-type calcium channels are important for the generation of repetitive activity in many neurons and other excitable cells (
Although T-channels in native cells share many properties, there is evidence for kinetic and pharmacological diversity. Notably, T-channels of thalamic relay neurons inactivate rapidly (
30 ms above -30 mV;
The cloning of three T-type calcium channels provided a basis for exploration of functional diversity among T-channels (1G and
1H) inactivate relatively rapidly (
15 ms above -30 mV), whereas the third (
1I) inactivates almost 10-fold more slowly (
1I is highly expressed in thalamic reticular neurons (
We recently examined in detail the gating kinetics of the 1G T-type calcium channel at the level of whole-cell currents (
We report here a study of the kinetics of activation and inactivation of the 1I T-type channel. The goal was to compare this slowly inactivating channel to the more typical
1G T-channel. Can gating of
1I be described by the same basic mechanism as
1G, but with different parameters (e.g., a slower inactivation rate), or are there fundamental differences in the way these two channels respond to voltage?
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MATERIALS AND METHODS |
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HEK293 cells stably expressing rat 1I (GenBank accession No.
AF086827) have been described previously (
1I reported by
1I clones ends with the sequence TGLHPSCWGMT, whereas the human
1I sequence reported by
1I results from alternative splicing of exon 33 to 34. The rat uses an internal splice site located 40 bp into exon 34, resulting in a change of the open reading frame. We have recently isolated a variant of rat
1I that splices these exons in the same manner as the human, resulting in a much longer open reading frame with considerable homology to the human sequence.
The stably transfected HEK293 cells were grown in cell culture as described previously (, measured before compensation (nominally 8095%). All experiments were performed at room temperature.
The holding potential was -100 mV. Except for some very long duration protocols (see Fig 9), both "raw" currents and online leak-subtracted (P/-4) currents were recorded, and only leak-subtracted data are shown. Data were usually sampled at 20 kHz (or 10 kHz for some inactivation protocols where tail currents were not analyzed in detail), after analogue filtering at 2550% of the sampling rate. It is noteworthy that some protocols required both high sampling rates and very long durations to accurately describe gating kinetics on different time scales (see Fig 4 and Fig 5 B). Data were analyzed primarily by Clampfit v. 8, although some analyses were done with Microsoft Excel or programs written locally in Visual Basic. Exponential fits were done with the Simplex or Levenberg-Marquardt methods of Clampfit v. 8, or (when parameters were constrained) using the Solver function of Excel to minimize the sum of squared errors. Values are given as means ± SEM, and for figures showing averaged data, error bars ± SEM are shown if larger than the symbol. Statistical significance levels given in the text are from paired two-tailed t tests (Excel), with P < 0.05 considered to be significant.
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Kinetic models were simulated using SCoP v. 3.51 (Simulation Resources). Simulated data were either converted to binary files and analyzed by Clampfit, or imported into Excel.
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RESULTS |
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Activation
Fig 1 illustrates the primary protocols used to examine the kinetics of 1I channels. Steps were given to voltages from -100 to +70 mV, either directly from -100 mV (Fig 1 A) or after activation of the channels by 10-ms steps to +60 mV (Fig 1 B). I-V relations measured from the two protocols are shown in Fig 1 C. Channel activation was clearly detectable by -60 mV, and the peak inward current was near -30 mV. The instantaneous I-V relationship (Fig 1 C, squares) was nonlinear, reflecting a high conductance to Ca2+ at negative voltages, a high conductance to Na+ at extreme positive voltages, but a lower conductance near the reversal potential. The reversal potential (+21.4 ± 1.3 mV, n = 5) corresponds to a Goldman-Hodgkin-Katz permeability ratio PCa/PNa = 95, compared with PCa/PNa = 150170 for
1G (
The two I-V relationships in Fig 1 C are the current at the time of peak activation at each voltage, and the instantaneous current after maximal activation (10 ms at +60 mV). At a given voltage, the driving force is the same, so the ratio of the two currents is the channel open probability (PO),* relative to that at +60 mV. We use this "relative PO" (PO,r) to assess the voltage dependence of channel activation (Fig 1 D). A fit of this activation curve to a single Boltzmann relation missed the data points at the foot of the curve (Fig 1 D, dashed curve), whereas a Boltzmann raised to the fourth power described the data somewhat more accurately (Fig 1 D, solid curve). The activation curves for 1G and
1I do not have precisely the same shape, but the point of 50% activation was
20 mV more positive for
1I.
The protocols of Fig 1 (A and B) were also used to measure the time dependence of channel gating. Fig 2 illustrates tail currents from Fig 1 B on a much faster time scale. Over the range of voltages where channels deactivate significantly, the tail currents were not well described by single exponentials, but the sum of two exponentials could fit the data well (Fig 2). Although only 10 ms is shown in Fig 2, the currents were fitted over the entire 50-ms pulse duration (Fig 1 B). The fitted time constants are shown in Fig 3. Especially at more negative voltages, the fast component was the largest (6272% of the total from -100 to -60 mV, with 2738% slow and 06% constant). Biexponential tails were also observed in the absence of Mg2+o, so this does not result from time-dependent block by Mg2+ (
We previously described 1G tail currents as monoexponential, but noted that fitting to the sum of two exponentials often yielded a small, fast component (
20%. In contrast to
1I (where the relative amplitude of the fast component was much larger), gating currents and/or Mg2+ block could account for much of the fast component of
1G. Thus, we conclude that a single exponential provides an adequate description of tail currents for
1G but not
1I.
The time constants at the most negative voltages should be interpreted with caution, as the fitted values varied significantly with the series resistance and clamp speed. For the tail current analysis in Fig 2 Fig 3, cells were selected for fast clamp, where the time to peak of the tail current was 0.35 ms or less. When the voltage clamp was slower, tail currents occasionally appeared to be monoexponential at -100 mV, as expected from RC filtering of the faster component by the electrode, but were still clearly biexponential at less negative voltages (-80 to -20 mV). The voltage-clamp error is largest at the most negative voltages, as the tail currents are larger and faster.
Envelope Test
The large, slowly inactivating currents observed at strongly depolarized voltages (Fig 1A and Fig B) look very different from classical T-currents, and could potentially reflect contaminating currents through channels endogenous to HEK 293 cells. To evaluate that, we used the "envelope test," where the current during a maintained depolarization is compared with the tail currents measured after voltage steps of different duration (Fig 4A and Fig B). The initial tail current amplitudes, scaled to account for the difference in driving force, superimpose upon the currents measured during depolarization, for steps to either -20 or +60 mV. Notably, the time course for inactivation was the same, measured either directly from the current during depolarization, or indirectly from the envelope of tail currents. This is consistent with both inward and outward currents flowing through the same population of channels. It also suggests that the large currents observed during long voltage steps used did not produce significant changes in ion concentrations, which can occur in small cells (
With the envelope protocol, tail currents at -100 mV were also described by the sum of two exponentials (Fig 4C and Fig D). As the pulse length increased, the time constants could be constrained to remain constant, but the relative amplitude of the slow component increased, from 0.12 ± 0.02 after 10 ms to 0.44 ± 0.05 after 1 s at +60 mV (n = 4; similar effect at -20 mV). Note that the two largest tail currents cross over in Fig 4 C. Slower tail currents after partial inactivation have been reported previously for 1I (
Activation, Deactivation, and Inactivation
Fig 3 summarizes several measurements of the time course of activation and inactivation, primarily from the protocols of Fig 1 (measurement of the time course of inactivation and recovery at more negative voltages is described below). One noteworthy feature is that macroscopic inactivation is much slower than activation or deactivation at extreme voltages, but at intermediate voltages (e.g., -40 mV), activation and inactivation kinetics are less clearly separated. We examined the time course of currents in this voltage region more thoroughly.
Upon repolarization, the open channel can either close (deactivate) or inactivate. Thus, the simplest interpretation of the biexponential decay of tail currents (Fig 2 Fig 3 Fig 4) is that the fast and slow components reflect deactivation and inactivation, respectively. We assessed that by comparing the time course of turn-on and turn-off of the current, measured at the same voltage (Fig 5). In principle, the time course of currents at any voltage should be described by the sum of exponentials, with the time constants depending only on voltage, and the relative amplitudes of the components depending also on the initial conditions. Thus, we constrained the time constants to be the same for the currents recorded as the channels turn on or turn off (
Upon weak depolarization, channels activated slowly after a clear delay, with time to peak as slow as 100 ms (Fig 3). The slow time course of activation was mirrored by the slow component of deactivation (Fig 5 A). Indeed, the time course of both activation and deactivation could be approximated by the sum of two exponentials, although the delay before activation was not well described (suggesting that the channel passes through more than two closed states before opening). The slower time constant was roughly similar to those found by fitting the tail current or the activation time course separately, and was considerably faster than the time constant of inactivation measured during long depolarization (110500 ms; Fig 3). This suggests that the two tail current time constants (measured during 50-ms steps) both primarily reflect the kinetics of the activation process.
In the voltage range where channels activate significantly, the biexponential fits to tail currents included a relatively large constant component (1625% from -50 to -20 mV; see also Fig 5 A). That is not realistic, as currents eventually inactivate almost completely in that voltage range (see Fig 7). This led us to examine currents during steps lasting several hundred milliseconds. That revealed a third exponential component, corresponding to inactivation (Fig 5 B). When the currents were fitted to the sum of three exponentials at each voltage, the time constants corresponded approximately to those found for fast deactivation, slow deactivation (or activation), and inactivation in Fig 3.
The three exponential components were most clearly demonstrated in tail currents at -40 mV (Fig 6), where the time constants were well separated, and the amplitudes were comparable and thus easily detectable. In seven cells, the time constants were 1.5 ± 0.2, 27 ± 4, and 118 ± 14 ms, with relative amplitudes 0.34 ± 0.04, 0.20 ± 0.01, and 0.43 ± 0.03, respectively (offset 0.026 ± 0.004), including three cells examined with no Mg2+o.
The time constants from these unconstrained three-exponential fits did not agree precisely with those from the simultaneous fits to turn-on and turn-off kinetics. In particular, the fast time constant was generally smaller for the fits to turn-off kinetics alone (compare Fig 6 with Fig 5 B, middle). By eye, the simultaneous fits missed part of the fast tail current, and underestimated the delay in activation. Also, in two out of four cells, the slow time constant at -50 mV was about threefold larger for the simultaneous fits (Fig 5 B, left). This suggests that a full description of 1I kinetics would require more than three exponential components in this voltage region. Our main conclusion from this analysis is that there are at least three components, with time constants on the 1-, 10-, and 100-ms time scales.
Complex tail current kinetics are not unexpected, as both deactivation and inactivation can contribute, on different time scales. However, the observation of bi- or triexponential tail currents for 1I contrasts sharply with
1G, where tail currents could be described well by a single exponential (
Inactivation
To examine the voltage dependence of inactivation, we first gave long depolarizations (1.5 s) to a range of voltages, followed by brief test pulses to -20 mV (Fig 7, protocol marked by two asterisks). The measured inactivation curve was well described by a single Boltzmann, with midpoint -64 mV. To determine whether that inactivation curve truly represented steady-state inactivation, we used the same protocol, except that channels were initially inactivated by 90% by a 0.6-s step to -20 mV. At steady state, the extent of inactivation should depend only on the conditioning voltage, and should be independent of the initial conditions. The data were again well described by a Boltzmann, but with a midpoint of -73 mV. This demonstrates that 1.5 s is not long enough to reach steady state, in contrast to our result with 1G (
To further explore the time and voltage dependence of inactivation, we compared the time course of inactivation and recovery at several voltages, using steps up to 3 s (Fig 8). Instead of achieving steady state, longer voltage steps revealed a second component of inactivation. When inactivation and recovery were fitted separately to single exponential functions at -70 or -80 mV, the time constant for recovery was significantly faster (Fig 3, open squares versus open diamonds). When inactivation and recovery were constrained to have the same time constant, single exponentials did not fit very well (Fig 8, dashed lines). Note that the single exponentials predict more rapid convergence of inactivation and recovery than observed at -70 mV. At -60 mV, where only recovery was tested with this protocol, there was a small but clear initial recovery phase, followed by subsequent development of additional inactivation. These results are not consistent with a single inactivation process, and suggest that voltage steps longer than 1 s begin to evoke a slow inactivation process. The nonmonotonic time course of recovery from inactivation at -60 mV could be explained if the slower inactivation process is more complete at that voltage.
Additional protocols were used to further demonstrate slow inactivation. Fig 9 A compares the time course of inactivation versus recovery at -70 mV for steps lasting up to 9 s. Inactivation continued to develop over several seconds, whereas recovery was maximal at 13 s. With the recovery protocol, channel availability at 9 s was 80 ± 5% of that at 3 s (n = 6; P = 0.008). Because of slow inactivation, even 9 s was not long enough to completely reach steady state, as currents measured at 9 s with the recovery protocol were 85 ± 3% of those measured with the inactivation protocol (n = 6; P = 0.005). The time course could be fitted to the sum of two exponentials, with = 0.73 ± 0.03 s and 4.7 ± 0.8 s, with 75 ± 5% inactivation (n = 6).
When test pulses were given while the holding potential was changed from -100 to -80 or -70 mV, two components of inactivation were again visible (Fig 9 C). Recovery from inactivation was essentially complete in the 2 s interval between the last pulse from -80 or -70 mV and the first test pulse from -100 mV.
Although these experiments do not fully characterize the kinetics of the slow inactivation process, they are sufficient to demonstrate its existence. For our purposes here, the main point is that the slow inactivation process interfered with direct measurement of steady-state inactivation for 1I.
State Dependence of Inactivation
For 1G, inactivation occurred primarily from open states at more depolarized voltages, but primarily from closed states near the midpoint of the inactivation curve (
1I (Fig 10). The amount of open-state inactivation was "predicted" (
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(1) |
where PI is the predicted probability that the channel will become inactivated, kI is the rate constant for inactivation (determined from the time constant for inactivation at depolarized voltages), and PO,r is calculated as in Fig 1 D for each data point. This calculation assumes that open-state inactivation is "absorbing" which would overestimate open-state inactivation and voltage-independent. Considerable inactivation occurred both at -80 mV, where there was no detectable channel opening and, thus, no predicted open-state inactivation, and at -70 mV, where channel opening was barely detectable (Fig 10). This demonstrates that inactivation can occur directly from closed states for 1I, and that closed-state inactivation dominates at the more negative voltages.
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The state dependence of inactivation at more depolarized voltages was examined with the protocols of Fig 1, including test pulses to +60 mV to assess inactivation (Fig 11). The recorded currents were converted to PO,r, to allow visual evaluation of the correlation between channel opening and inactivation. 500-ms voltage steps to -60 or -50 mV opened few channels, but induced considerable inactivation (Fig 11 A). Steps to +60 mV produced about twice as much integrated PO,r than steps to -30 mV, but the extent of inactivation was comparable (Fig 11A and Fig B). This suggests that about half of the inactivation at -30 mV must have occurred from closed states. The measured inactivation exceeds the predicted open-state inactivation from -90 to -10 mV (Fig 11 B).
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Closed-state inactivation can be demonstrated even for relatively brief depolarizations with the protocol of Fig 1 B (see also Fig 11C and Fig D). With an interpulse voltage of -100 mV, two 10-ms steps to +60 mV separated by 70 ms produced 10 ± 1% inactivation (n = 5). Considerably more inactivation occurred at -60 mV (21 ± 2%), even though the integrated PO,r was only slightly greater. PO,r is clearly lower at -20 mV than at +60 mV, because a significant fraction of channels deactivate at -20 mV (Fig 11 B), as expected from the activation curve (Fig 1 D). However, the extent of inactivation was indistinguishable at -20 and +60 mV (37 ± 2% vs. 39 ± 2%, respectively). This implies that closed-state inactivation is significant even at -20 mV. Furthermore, the net rate of inactivation must be the same at -20 and +60 mV, despite the difference in PO,r. That is also shown by the time constants for inactivation, which are essentially constant above -30 mV (Fig 3). Thus, certain closed states, presumably those close to the open state, inactivate as rapidly as the open state itself.
Completeness of Inactivation
Inactivation of 1I is incomplete, on the time scale examined. For the longest steps given with the envelope protocol (Fig 4), PO,r after 1 s at -20 mV was 0.021 ± 0.006 (measured at the end of the depolarizing step) or 0.036 ± 0.008 (from the tail currents); at +60 mV, corresponding values were 0.032 ± 0.006 and 0.033 ± 0.007 (n = 4). From single exponential fits to those records, the time constants for inactivation were 165 ± 14 ms at +60 mV, and 167 ± 16 ms at -20 mV, with offsets of 0.032 ± 0.005 and 0.023 ± 0.005, respectively. If inactivation occurred at the measured rate and was fully absorbing, the residual PO,r at 1 s would be only 0.003 ± 0.001 at +60 mV and 0.003 ± 0.002 at -20 mV. Thus, fast inactivation spares
2% of
1I channels at steady state, over a wide voltage range, as previously observed for
1G (
= 130 ms and an offset of PO,r = 0.05 (on average,
= 134 ± 9 ms with offset 0.037 ± 0.005, n = 5). That caused the predicted inactivation to exceed 1.0 at the most depolarized voltages (Fig 11 B).
Kinetic Model
We described gating of 1I using the same kinetic scheme proposed for
1G (Fig 12 A and Table 1). Briefly, the model assumes a linear pathway for activation (voltage sensor movement followed by channel opening), with inactivation allosterically coupled to the first three voltage sensors to activate (
1G. The inactivation rate (kI) was determined from the time constant for inactivation at depolarized voltages (Fig 3), and the reverse rate (k-I) from the extent of inactivation. Given those values, the allosteric factor for recovery (h) fixed the limiting rate of recovery from inactivation at negative voltages. The channel opening (kO) rate limits the rate of activation at positive voltages. The charge movement associated with voltage sensor movement (and with channel closing) was kept the same as for
1G. The other four parameters (kV, k-V, k-O, and f) were not directly determined, and were adjusted to describe the activation curve (Fig 12 B), inactivation curve (Fig 12 C), activation and deactivation time constants, and current time to peak (Fig 3). The kinetics of activation for weak depolarizations (Fig 12 D) was strongly affected by the speed of voltage sensor movement, such that twofold changes in kV and k-V produced activation that was clearly too fast or too slow. The model also produced a considerable amount of closed-state inactivation. The measured inactivation exceeded the predicted open-state inactivation (Fig 13), much as observed experimentally (Fig 11).
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Table 1 shows that most rates are 512-fold slower for 1I, compared with
1G. But the kinetics of
1I are not simply scaled with respect to
1G, since one crucial rate is actually fourfold faster for
1I (channel closing, k-O). That has several consequences for channel gating, some of which are not intuitively obvious. For
1I, the C4-O equilibrium favors C4 at -60 mV and below, and, even at more positive voltages, a significant fraction of channels remain closed. This contributes to the positive shift in the voltage dependence of activation for
1I. It also allows more closed-state inactivation, since the C4 and C3 states are assumed to inactivate as rapidly as O.
How does the model produce the three exponential components of tail currents? At -40 mV, channel closing is much faster than inactivation (k-O = 324 s-1 and kI = 7 s-1), and the C-C rates are intermediate (14160 s-1). Both the C-O equilibrium, and the equilibrium for activation of an individual voltage sensor, favor activation slightly. During a tail current, a fraction of the channels move rapidly from O to C4; next, slower C-C transitions pull more channels away from the open state; finally, inactivation pulls the remaining channels away from the open and closed states. A large fraction of channels transiently accumulates in closed states, but at steady state, nearly all of the channels are inactivated. On a 500-ms time scale, three exponentials were required to fit tail currents simulated from the model at -40 mV ( = 1.3, 23, and 160 ms, with nearly equal amplitudes). The three time constants correspond closely to the kinetics of the O-C, C-C, and C-I transitions.
Why are three exponential components not apparent for 1G tail currents? First, the channel closing rate is much closer to the inactivation rate (at -40 mV k-O = 81 s-1 and kI = 70 s-1), so closing and inactivation occur on the same time scale. In addition, the C-O equilibrium is strongly to the right for
1G, by about a factor of 50 at -40 mV. This means that most channels that inactivate do so directly from the open state (
1I channels tend to close first). The C-O equilibrium also makes the net rate of channel closing much slower than k-O. As a result, with the parameters used for
1G, the model predicts tail currents that are described almost perfectly by single exponentials. This can be attributed entirely to the difference in channel closing rate between
1G and
1I, relative to the other rate constants in the models. For
1I, when k-O at 0 mV was changed from 100 to 3 s-1 (eightfold slower than
1G, versus fourfold faster), tail currents were effectively monoexponential over a wide voltage range (unpublished data).
Some features of the data were not described well by the model for 1I. In particular, there was no slow component of inactivation. Presumably, that reflects a separate slow inactivation mechanism, which is not included in the model. Thus, the model should be considered to apply primarily to
1I kinetics on the <1-s time scale. In addition, although the model produced clearly biexponential tail currents on a 50-ms time scale (Fig 12 D), and the two time constants were close to the experimental values (Fig 3), the relative amplitudes of the two components were not well reproduced (note the large amplitude of slow deactivation at -60 mV in Fig 13 C versus Fig 11 C). Also, the model did not predict the change in relative amplitudes of fast and slow deactivation with partial inactivation (Fig 4C and Fig D), so that effect could result from a kinetic process not included in the model (e.g., facilitation;
1I, rather than as a complete quantitative description.
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DISCUSSION |
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Our goal was to compare the gating kinetics of the 1I calcium channel to
1G, which exhibits more "typical" T-channel kinetics, using whole-cell recording and kinetic modeling. The starting point was the observation that
1I activates and inactivates more slowly than
1G. Mechanistically, those two differences could be explained either by slower microscopic inactivation or by slower microscopic activation. If activation is slow and rate-limiting for macroscopic inactivation (
To fully explain our results, we found that both activation and inactivation had to be slower for 1I. Slower activation is not sufficient, as inactivation becomes voltage-independent above -50 mV, suggesting that microscopic inactivation is rate-limiting. Note also that the activation rate and the time to peak accelerate at least 10-fold with depolarization above -50 mV, which is further evidence that activation is not rate-limiting for inactivation in that region. On the other hand, if only microscopic inactivation is slowed, the slow activation and deactivation kinetics observed near -40 mV (Fig 5 A) would not occur. However, gating of
1I is not uniformly slower than
1G, since deactivation is faster for
1I at strongly hyperpolarized voltages (Fig 3). A quantitative difference in the channel closing rate produced qualitative differences in kinetic behavior, most notably multiexponential tail currents for
1I.
Gating Kinetics of 1I
Several basic features of gating of 1I agree with previous studies. Compared with
1G and most other T-channels, activation and inactivation are slow, but deactivation is fast (
We find a more positive activation curve (V1/2 = -20 mV) than most previous studies ([-44 mV] 1I channels continue to activate at positive potentials (Fig 1 D), whereas most previous studies of activation were based primarily on data at negative voltages. If we use chord conductances rather than PO,r, and fit only to +10 mV, a single Boltzmann gives an apparent V1/2 = -43 mV. The chord conductances are affected not only by the channel open probability, but also by the voltage dependence of the open-channel current, so that V1/2 does not accurately describe the voltage dependence of channel opening.
Although we were unable to reach steady-state inactivation without evoking slow inactivation, we would estimate from Fig 7 that fast inactivation is half complete near -70 mV. This agrees well with several previous studies (1I using 15-s pulses. The kinetic differences observed by
1I clone (as described in MATERIALS AND METHODS).
Some kinetic parameters varied significantly among cells. For example, in one set of 16 cells, the time constant for inactivation during 600-ms steps to -20 mV was 147 ± 9 ms (SD = 37 ms). In cells tested at both -20 and +60 mV, inactivation rates were similar, so the variability does not result from the voltage dependence of inactivation. Inactivation time constants could vary twofold for cells recorded on the same day, with no obvious explanation. Such variability could contribute to the quantitative differences among previous studies of 1I kinetics, and might also indicate that
1I gating can be regulated by, as yet, unknown factors.
One striking difference from previous studies on 1I is our observation that tail currents are bi- or even triexponential. It is not clear why this was not observed previously, although we appear to have examined a wider voltage range and longer time scale than in some other studies.
The kinetics of 1I are similar in several respects to the T-current of thalamic reticular neurons (
10 mV to more positive voltages, compared with the more classical T-current of thalamic relay neurons. Combined with the high level of expression of
1I in thalamic reticular neurons (
1I makes a major contribution to the T-current in those cells.
Gating Mechanisms of 1I
Activation and inactivation of 1I can be described by an allosteric model, where voltage-dependent activation is coupled to an intrinsically voltage-independent inactivation process (Fig 12 Fig 13). The same model, but with substantially different parameters (Table 1), was previously used to describe
1G (
Several features of gating of 1I do qualitatively resemble
1G. Inactivation is strong, but only 9899% complete, even with strong depolarization. The macroscopic time constants for inactivation and recovery reach limiting rates at positive and negative voltages, respectively. It is especially striking that inactivation is nearly voltage-independent over a wide range (-30 to +70 mV) for both channels. Only in a narrow voltage region do the time constants for inactivation and recovery exhibit the voltage dependence expected from coupling to the activation process (Fig 3). This appears to result from a relatively weak allosteric coupling, such that the inactivation and recovery rates do not vary dramatically with the state of the channel, although the coupling is strong enough to produce the observed voltage dependence of inactivation. A similar point was made recently by
For both 1G and
1I, our models assume that inactivation is as fast from certain closed states (Fig 12 A, C3 and C4) as from the open state itself. For
1G, that assumption was made merely to improve the quality of the fit to the data. For
1I, we present more direct evidence: inactivation is as fast at -20 mV as at +60 mV, even though PO,r is considerably lower at -20 mV (Fig 1 D versus Fig 3; Fig 11C and Fig D). In retrospect, that argument also holds for
1G, since the inactivation rate reached a limiting value by -30 mV, where only 70% of the channels were activated (
1I clearly differs from
1G in the absolute rates of activation and inactivation. More significantly, there were two apparent qualitative differences between the two channels, with
1G exhibiting monoexponential tail currents and less closed-state inactivation at positive voltages (
1I. In our model, that rate is fourfold faster for
1I in absolute terms, and 2050-fold faster relative to the other rate constants in the model.
Slow inactivation on a time scale of seconds or minutes appears to be a nearly universal property of channels that also exhibit a fast inactivation process. We find slow inactivation for 1I on the 110-s time scale (Fig 9), and a preliminary report has appeared of slow inactivation for
1G (
1G (
The molecular basis of T-channel inactivation remains to be determined. Some features resemble Na+ channels more closely than other Ca2+ channels; at the level of amino acid sequence, T-channels are only slightly closer to other Ca2+ channels than to Na+ channels. But there is no clear homology between T-channels and Na+ channels in the III-IV linker, which is critical for inactivation of Na+ channels. It will also be important to find the molecular determinants of the differences in activation and inactivation kinetics among T-type channels.
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Footnotes |
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The present address of C.J. Frazier is Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610.
* Abbreviations used in this paper: PO, open channel probability; PO,r, PO relative to that produced by a 10-ms depolarization from -100 to +60 mV.
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Acknowledgements |
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This work was supported in part by National Institutes of Health grants (NS24471) to S.W. Jones and (NS38691) E. Perez-Reyes, an NIH postdoctoral fellowship (NS10828) to C.J. Frazier, and a Howard Hughes Medical Institute grant to Case Western Reserve University School of Medicine.
Submitted: 17 July 2001
Revised: 4 September 2001
Accepted: 4 September 2001
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