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I. The Slow and the Fast Gating Modes and their Modulation by ß Subunits

Siro Luvisetto¹, Tommaso Fellin¹, Michele Spagnolo¹, Bruno Hivert^1,2, Paul F. Brust^3,4, Michael M. Harpold³, Kenneth A. Stauderman^3,5, Mark E. Williams^3,6, and Daniela Pietrobon¹

¹ Department of Biomedical Sciences and Consiglio Nazionale delle Ricerche Institute of Neuroscience, University of Padova, 35121 Padova, Italy
² Department of Biology, Faculte des Sciences de Luminy, 13288 Marseille, cedex 9, France
³ SIBIA Neurosciences, La Jolla, CA 92037
⁴ Senomyx Inc., La Jolla, CA 92037
⁵ Neurogenetics Inc., La Jolla, CA 92037
⁶ Merck Research Labs, San Diego, CA 92121

Address correspondence to Daniela Pietrobon, Dept. of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy. Fax: 39-049-8276049. email: daniela.pietrobon{at}unipd.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The single channel gating properties of human Ca_V2.1 (P/Q-type) calcium channels and their modulation by the auxiliary ß_1b, ß_2e, ß_3a, and ß_4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human Ca_V2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463–474). Here, we report the characterization of two modes of gating of human Ca_V2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). Ca_V2.1 channels containing any of the four ß subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, Ca_V2.1 channels display different rates of inactivation and different steady-state inactivation depending on the ß subtype. The type of ß subunit also modulates the relative occurrence of the slow and the fast gating mode of Ca_V2.1 channels; ß_3a promotes the fast mode, whereas ß_4a promotes the slow mode. The prevailing mode of gating of Ca_V2.1 channels lacking a ß subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.

Key Words: Ca²⁺ channel • gating mode • synaptic transmission • familial hemiplegic migraine • auxiliary subunit

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Voltage-gated P/Q-type calcium channels (Ca_V2.1) are located in presynaptic terminals and somatodendritic membranes throughout the brain (Volsen et al., 1995; Westenbroek et al., 1995), and have a prominent role in controlling neurotransmitter release (Dunlap et al., 1995). The somatodendritic localization of Ca_V2.1 channels points to additional postsynaptic roles in, for example, neural excitability (Bayliss et al., 1997; Pineda et al., 1998; Mori et al., 2000), synaptic integration and plasticity (Eilers et al., 1996; Magee et al., 1998), and gene expression (Sutton et al., 1999). The importance of Ca_V2.1 channels in brain function is stressed by the evidence that mutations in CACNA1A, the gene encoding Ca_V2.1₁ subunits, cause a group of dominantly inherited human neurological disorders, including familial hemiplegic migraine, episodic ataxia type-2, and spinocerebellar ataxia type 6 (Ophoff et al., 1996; Zhuchenko et al., 1997). Mutations at the mouse orthologue cause a group of recessive neurological disorders, including the tottering, leaner, and rocker phenotypes with ataxia and absence epilepsy, and the rolling Nagoya phenotype with ataxia without seizures (Fletcher et al., 1996; Pietrobon, 2002). In the lethargic mouse, a mutation in the gene encoding the auxiliary calcium channel ß₄ subunit causes a clinical phenotype very similar to that of tottering (Burgess et al., 1997). Knockout mice with a null mutation in the Ca_V2.1₁ gene show severe cerebellar ataxia and dystonia and selective progressive cerebellar degeneration (Jun et al., 1999; Fletcher et al., 2001).

The single channel gating properties of Ca_V2.1 channels are critical determinants of the time course and magnitude of the various Ca²⁺-dependent processes they control. Both experiments and simulations have shown that even small changes in the kinetics of channel opening and/or closing and in channel open probability can strongly affect the time course and magnitude of Ca²⁺ influx during an action potential (McCobb and Beam, 1991; Borst and Sakmann, 1998; Sabatini and Regehr, 1999; Colecraft et al., 2001; Bischofberger et al., 2002; Meinrenken et al., 2002, 2003). Given the steep dependence of neurotransmitter release on Ca²⁺ influx (Dodge and Rahamimoff, 1967; Bollmann et al., 2000; Schneggenburger and Neher, 2000), the detailed gating properties of single Ca_V2.1 channels will have a strong impact especially on the time course and magnitude of neurotransmitter release at central synapses, where P/Q channels appear to be more effectively coupled to release than other Ca²⁺ channel types (Mintz et al., 1995; Wu et al., 1999; Qian and Noebels, 2001). Despite the critical role of Ca_V2.1 channel gating in determining neurotransmission efficacy at central synapses, surprisingly few data are available on the single channel gating properties of native P/Q-type Ca²⁺ channels (Usowicz et al., 1992; Forti et al., 1994; Tottene et al., 1996) or recombinant Ca_V2.1 channels (Yatani et al., 1994; Bourinet et al., 1999; Hans et al., 1999; Colecraft et al., 2001; Tottene et al., 2002). The discovery that mutations causing familial hemiplegic migraine increase the open probability and the single channel Ca²⁺ influx through human Ca_V2.1 channels (Hans et al., 1999; Tottene et al., 2002) and, as a consequence, facilitate the induction and the propagation of cortical spreading depression (van den Maagdenberg et al., 2004) fosters the interest in extending our knowledge of the single channel properties of human Ca_V2.1 channels. Therefore, one of our aims here was to obtain a detailed characterization of the gating properties of human Ca_V2.1 channels at the single channel level.

In heterologous expression systems, ß subunits are powerful regulators of both channel activity and number of channels expressed in the membrane (Walker et al., 1998; Dolphin, 2003a). In particular, for the Ca_V2.1 channel, there is evidence for both a chaperone-like effect of ß subunits on channel trafficking to the plasma membrane (Brice et al., 1997; Bichet et al., 2000) and modulation of the voltage range of activation and inactivation as well as the kinetics of inactivation of the whole-cell current (Stea et al., 1994; De Waard and Campbell, 1995; De Waard et al., 1995; Cens et al., 1996). Four different genes encode ß subunits (ß_1,2,3,4) that are differentially expressed in different neurons and during development (Tanaka et al., 1995; Witcher et al., 1995; Ludwig et al., 1997; Volsen et al., 1997; Vance et al., 1998; Burgess et al., 1999). Regional differences in brain expression pattern can also occur for splice variants of the same ß subunit (Helton et al., 2002). Native P/Q-type channels can contain each of the four different ß subunits (Liu et al., 1996), and the fractional contribution of a particular ß subunit for channel formation varies among different brain regions (Pichler et al., 1997). Different combinations of a Ca_V2.1₁ subunit with auxiliary ß subunits most likely contribute to generate the large functional diversity of native P/Q-type calcium channels (Mintz et al., 1992; Usowicz et al., 1992; Randall and Tsien, 1995; Tottene et al., 1996; Forsythe et al., 1998; Mermelstein et al., 1999). Whole-cell current recordings in heterologous expression systems revealed quite different kinetics of inactivation and slightly different voltage ranges of activation depending on the ß subtype combined with the Ca_V2.1₁ subunit (Sather et al., 1993; Stea et al., 1994; De Waard and Campbell, 1995; De Waard et al., 1995; Cens et al., 1996; Moreno et al., 1997; Krovetz et al., 2000; Restituito et al., 2000; Sokolov et al., 2000; Sandoz et al., 2001; Helton and Horne, 2002; Helton et al., 2002; Tsunemi et al., 2002). However, the effect of different ß subunits on the single channel gating properties of Ca_V2.1 channels (and also of the other Ca_V channels, with the exception of Ca_V1.2) remains unknown. Interestingly, distinct ß subunits confer unique single channel gating properties to L-type channels extending well beyond differences in inactivation (Colecraft et al., 2002). The regulation of Ca_V2.1 channel properties by variations in ß subunit composition appears as a potential mechanism for tuning channel behavior to support a given physiological role, and might contribute to create the great diversity of release efficacy and short-term synaptic plasticity at different synapses (Atwood and Karunanithi, 2002). Therefore a second aim here is to study how the presence of different auxiliary ß subunits in the human Ca_V2.1 channel alters gating at the single channel level.

Single channel patch-clamp recordings on human embryonic kidney HEK293 cells expressing human Ca_V2.1 channels revealed a complex modal gating of these channels, which is described in this and the accompanying paper (Fellin et al., 2004). Here, we report the characterization of two modes of gating of human Ca_V2.1 channels, the slow mode and the fast mode, that differ in mean closed times and latency to first opening, in voltage dependence of the open probability, in kinetics of inactivation, and voltage dependence of steady-state inactivation. The study of Ca_V2.1 channels containing four different ß subunits shows that both the relative occurrence of the slow and fast gating modes and the inactivation properties within each mode are modulated by the type of ß subunit. We also show that the prevailing mode of gating of Ca_V2.1 channels lacking a ß subunit is a low-p_o mode different from both the fast and the slow gating modes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture and Transfection
HEK293 cells (American Type Culture Collection, CRL-1533) were stably transfected with cDNA constructs encoding the human Ca_V2.1₁ (_1A-2), _2b-1, and ß_1b (A68-90 cell line) or ß_2e (E1H2 cell line) or ß_3a (PB1-14 cell line) or ß_4a (10–13 cell line) subunits using a standard calcium phosphate–mediated procedure (Hans et al., 1999). Antibiotic-resistant colonies were selected in medium consisting of Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 6% bovine calf serum, penicillin, streptomycin, and G418 or a combination of G418 and Zeocin depending on the constructs used. Cells were maintained on standard tissue culture plates at 37°C and 5% CO₂. Parental lines and subsequent subclones were selected based on the following criteria: (a) functional responses in a fluo3 dye–based assay where increases in intracellular Ca²⁺ are measured in response to KCl depolarization; (b) Northern and Western analysis, and (c) electrophysiological characterization. All cell lines were incubated at 28°C for 12–24 h before electrophysiological measurements (and in the case of the PB1-14 cell line also before the fluo3 assay) (Hans et al., 1999).

In the case of transient transfection of HEK293 cells with cDNA constructs encoding the human Ca_V2.1₁ (_1A-2) and _2b-1 subunits (or of PB1-14 cells with cDNA constructs encoding the ß_3a subunit), CD4 expression plasmids were included to permit the identification of transfected cells as in Hans et al. (1999).

In untransfected HEK293 cells, neither Ca_V2.1₁ nor ß transcripts were detected by Northern blots and neither Ca_V2.1₁ nor ß protein was detected on Western blots (not depicted). In contrast, both Northern and Western blot assays revealed the presence of an endogenous _2b-1 subunit in the same cells (not depicted).

Patch-clamp Recordings and Data Analysis
Single channel patch-clamp recordings were performed following standard techniques, as in Hans et al. (1999). Currents were recorded at room temperature with a DAGAN 3900 patch-clamp amplifier, low-pass filtered at 1 kHz (–3 dB, 8-pole Bessel filter), sampled at 5 kHz, and stored for later analysis on a PDP-11/73 computer. All single channel recordings were obtained in the cell-attached configuration. The pipette solution contained (in mM) 90 BaCl₂, 10 TEA-Cl, 15 CsCl, 10 HEPES (pH 7.4 with TEA-OH). The bath solution contained (in mM) 140 K-gluconate, 5 EGTA, 35 l-glucose, 10 HEPES (pH 7.4 with KOH). The high-potassium bath solution was used to zero the membrane potential outside the patch. Liquid junction potential at the pipette tip was +12 mV, and this value should be subtracted from all voltages to obtain correct values of membrane potentials.

Linear leak and capacitative currents were digitally subtracted from all records used for analysis. Open-channel current amplitudes were measured by manually fitting cursors to well-resolved channel openings. Values at each voltage are averages of many measurements. Open probability, p_o, was computed by measuring the average current, I, in a given single channel current record and dividing it by the unitary single channel current, i. To obtain activation curves, p_o values were calculated in patches containing only one channel by averaging the open probabilities measured in each sweep with activity at a given voltage (excluding the last shut time). Single channel activation curves were best fitted with the Boltzmann equation p_o = p_omax x {1 + exp[–(V – V_1/2)/k]}^–1, where k = RT/zF. For open and closed time histograms, a channel opening or closure was detected when more than one sampling point crossed a discriminator line at 50% of the elementary current. Histograms of open and closed times were fitted with sums of decaying exponentials. Log binning and fitting of the binned distributions were done as described by McManus et al. (1987) and Sigworth and Sine (1987). The first closed time was used to generate the cumulative first latency histogram. The best fit was determined by the maximum likelihood maximization (Colquhoun and Sigworth, 1983), and the best minimum number of exponential components was determined by the maximum likelihood ratio test (Rao, 1973). All values are given as mean ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell-attached single channel recordings on HEK293 cells stably coexpressing human Ca_V2.1₁ (_1A-2), ß_1b, and _2b-1 subunits revealed that, in many patches containing only one channel, the same channel showed two different modes of gating. Fig. 1 A shows consecutive traces from a single channel patch displaying activity of the same Ca_V2.1 channel in two different periods at the same voltage (+30 mV). Analysis of the open and closed time histograms in the two periods shows that the two modes of gating differ mainly in the intervals of time spent by the channel in closed states (Fig. 1 B and Fig. 2 A). We have called these two modes of gating "slow" and "fast" for reasons that will become clear below, when the kinetics of inactivation and the latencies to first opening of a Ca_V2.1 channel in the two modes will be considered.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. A single human CaV2.1 channel shows two different modes of gating: the fast and slow modes. Cell-attached single channel recordings with 90 mM Ba2+ as charge carrier on HEK293 cells stably coexpressing human CaV2.11 (1A-2), ß1b and 2b-1 subunits. Depolarizations were 720 ms long and were delivered every 4 s from a holding potential of –80 mV. Records were sampled and filtered at 5 and 1 kHz, respectively. (A) Consecutive traces in two different periods from a patch containing a single CaV2.1 channel. By visual inspection, the channel was in the mode of gating shown on the left (the slow gating mode) during the first 8 min of recording, and in the mode of gating shown on the right (the fast gating mode) during the remaining 40 min. Single channel current and conductance were identical in the two periods. (B) Log–log plots of the open and closed time distributions of the same single channel in the two periods in fast and slow gating mode. The dark solid line in each plot is the best-fitting sum of two or three exponential components for the open or closed times, respectively (each exponential component is shown as a dotted line); time constants of the open times: 0.54 and 1.42 ms (relative areas 40 and 60%) for the slow mode and 0.40 and 1.36 ms (relative areas 71 and 29%) for the fast mode; time constants of the closed times: 0.34, 3.17, and 13.4 ms (relative areas 64, 14, and 22%) for the slow mode, and 0.29, 1.17, and 3.54 ms (relative areas 43, 38, and 19%) for the fast mode. (C) Open probability, po, as a function of voltage for the same single channel gating in the fast (empty symbol) and slow (dark symbol) mode. Fit of the two activation curves with a Boltzmann equation gives V1/2 = 34 mV, k = 4.5 mV, and po max = 0.64 for the slow mode and V1/2 = 29 mV, k = 6.6 mV, and po max = 0.67 for the fast mode. The companion paper (Fellin et al., 2004) describes two additional modes of gating of CaV2.1 channels named b and nb modes; the single channel shown here was in the nb mode for the entire duration of the recording.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. A single human CaV2.1 channel in the slow gating mode spends longer intervals of time in closed states and activates at more positive voltages than in the fast gating mode. Single channel recordings as in Fig. 1 on HEK293 cells stably coexpressing human CaV2.11 (1A-2), 2b-1, and ß1b, ß2e, or ß4a subunits. (A) Time constants (open and closed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels (1A-2–ß1b–2b-1) in the slow (dark bar) and the fast (empty bar) gating mode. Average values were obtained from single channel patches (n = 5 for both the slow and fast mode) in which, by visual inspection, the channel showed only (or mainly) one gating mode or two clearly separated periods in the two modes. Average single channel current and conductance were identical for the five channels in either the slow or fast gating mode. Statistical significance of difference between paired values using student t test: *, P < 0.05; **, P < 0.001. (B) Normalized open probability, po, as a function of voltage of single CaV2.1 channels containing different ß subunits gating in the fast (empty symbol) and the slow mode (dark symbol). Average values were obtained from single channel patches in which the channel showed only (or mainly) one gating mode or two clearly separated periods in the two modes: n = 5, 5, and 6 for the slow mode and n = 5, 4, and 4 for the fast mode of channels containing the ß1b, ß2e, and ß4a subunits, respectively.

In both gating modes, open and closed time histograms were best fit by the sum of two and three exponential components, respectively. Both the time constants and the relative areas of the two exponential components best fitting the open time histograms were similar for the two gating modes, whereas two of the closed time constants (_c2 and _c3) were significantly larger in the slow mode than in the fast mode (Fig. 1 B and Fig. 2 A). Moreover, their relative areas were different in the two gating modes, showing a larger contribution of the longest closed time intervals in the slow with respect to the fast gating mode. As a result, at +30 mV, the open probability of the channel in the fast gating mode was about twice that in the slow mode (Fig. 1 C and Fig. 2 B). Measurements of the open probability, p_o, as a function of voltage revealed that the Ca_V2.1 channel activates at more negative voltages in the fast gating mode than in the slow mode (Fig. 1 C and Fig. 2 B). Fit with a Boltzmann function of the average activation curve of Ca_V2.1 channels containing the ß_1b subunit gave V_1/2 values of 25 and 32 mV (with k values of 5.3 and 4.2 mV) for the fast and slow gating mode, respectively. The single channel currents and conductance were identical in the two gating modes (not depicted, c.f. Table I).

Single channels containing the different ß subunits in either the fast or the slow gating mode had similar open and closed time histograms at +30 mV, as shown by the average values of the open and closed time constants in Table I. Only the channels with the ß_2e subunit displayed some significant difference with respect to those with the ß_1b subunit, and, precisely, both _c2 and _c3 in the slow gating mode were larger (P < 0.05). As a consequence, the open probability at +30 mV in the slow gating mode was smaller with the ß_2e than with the ß_1b subunit (P < 0.05).

Overall, from the data in Table I and Fig. 2, we can conclude that channels containing different ß subunits in either the fast or the slow gating mode show only minor differences in the rate constants of the transitions between closed and open states within each gating mode.

In addition to different properties of activation, the human Ca_V2.1 channel showed different properties of inactivation in the two modes of gating (Figs. 3 and 4). During depolarizations to +30 mV, lasting 720 ms, single channels containing the ß_4a subunit showed both inactivating and noninactivating activity in the fast gating mode, but almost exclusively noninactivating activity in the slow mode (Fig. 3 A). Thus, as shown by the average single channel ensemble currents, inactivation was more rapid in the fast than in the slow gating mode. The kinetics of inactivation were more rapid in the fast than in the slow gating mode independently of the type of ß subunit combined with the Ca_V2.1₁ subunit (Fig. 3 B). However, the kinetics of inactivation in each gating mode were different depending on the type of ß subunit. Judging from the fraction of peak current remaining at the end of the test pulse, the most rapidly and slowly inactivating channels in the fast gating mode were those containing the ß_4a and the ß_2e subunit, respectively (with intermediate similar values with ß_3a and ß_1b; c.f. also average fraction of inactivating traces in Table II). Judging from the time constant of inactivation, _i, obtained from fitting the ensemble averages of the inactivating sweeps (Table II), Ca_V2.1 channels containing the ß_4a subunit inactivated more slowly than those containing the ß_3a or the ß_1b subunit. The discrepancy may arise in part from the existence and different contribution of an additional noninactivating gating mode of Ca_V2.1 channels, the b mode, that will be described in the companion paper (Fellin et al., 2004). The different kinetics of inactivation of channels with different ß subunits in the slow gating mode could be best appreciated at voltages >30 mV (not depicted).

View larger version (40K): [in this window] [in a new window]   FIGURE 3. A single human CaV2.1 channel in the fast gating mode inactivates more rapidly than in the slow gating mode, independently of the ß subtype. Single channel recordings as in Fig. 1 on HEK293 cells stably coexpressing human CaV2.11 (1A-2), 2b-1, and ß4a, ß1b, ß2e, and ß3a subunits. (A) Representative traces at +30 mV from two patches containing a single CaV2.1 (1A-2–ß4a–2b-1) channel in either the fast (left) or the slow gating mode (right), and pooled average single channel ensemble currents at +30 mV from five (n= 323 traces) and four patches (n = 213 traces) containing a single channel in the fast and slow gating mode, respectively. (B) Pooled average single channel ensemble currents at +30 mV of CaV2.1 channels containing different ß subunits gating in either the fast (left) or slow mode (right). Fast mode: ß1b, n = 133 from three patches; ß2e, n = 301 from four patches; ß3a, n = 201 from five patches. Slow mode: ß1b, n = 139 from three patches; ß2e, n = 163 from three patches. Average ensemble currents were obtained from single channel patches in which, by visual inspection, the channel showed only (or mainly) one gating mode or two clearly separated periods in the two modes. Most of the data for CaV2.1 channels containing the ß3a subunit were obtained from PB1-14 cells transfected with ß3a cDNA. The fraction of peak current remaining after 720 ms was 49, 72, 86, and 66% for channels in the fast mode with ß4a, ß1b, ß2e, and ß3a, respectively, and 89, 100, and 100% for channels in slow mode with ß4a, ß1b, and ß2e, respectively.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Steady-state inactivation of human CaV2.1 channels occurs at more negative voltages in the fast than in the slow gating mode, independently of the ß subtype. Single channel recordings as in Fig. 1 on HEK293 cells stably coexpressing human CaV2.11 (1A-2), 2b-1, and ß1b, ß2e, ß3a, or ß4a subunits. (A) Open probability in successive depolarizations at +30 mV as a function of time during recordings in which the holding potential was changed as indicated above the horizontal bars, from two representative patches containing a single CaV2.1 (1A-2–ß4a–2b-1) channel in either the fast (left) or the slow gating mode (right). (B) Normalized open probability, (po)n, at +30 mV of CaV2.1 channels gating in the fast and slow modes containing either the ß1b or the ß4a subunits as a function of holding potential, Vh, obtained from patches containing a single channel showing only, or mainly, one gating mode or two clearly separated periods in the two modes. Po at different holding potentials was obtained by averaging the open probability of all consecutive sweeps at a given Vh in experiments (n = 3–8) like those in A. In each experiment, the open probability was normalized to the value at Vh = –80 mV. (C) Normalized open probability, (po)n, at +30 mV of CaV2.1 channels containing different ß subunits all gating in the fast mode as a function of Vh. ß1b (), ß2e (), ß3a (), ß4a (). Most of the patches contained a single channel. Part of the data for CaV2.1 channels containing the ß2e subunit were obtained from HEK293 cells transiently coexpressing human CaV2.11, 2b-1, and ß2e subunits. Most of the data for CaV2.1 channels containing the ß3a subunit were obtained from PB1-14 cells transfected with ß3a cDNA.

The relative occurrence of the slow and fast modes of gating of Ca_V2.1 channels appeared to depend on the type of ß subunit. The fraction of time spent by single recombinant Ca_V2.1 channels in each gating mode was evaluated in patches containing only one channel, whose activity could be recorded for at least 12 min. In each patch, only the first 12 to 16 min of single channel activity were considered, to avoid possible distortions due to different durations of the recording in different patches. Moreover, due to the difficulty of separating with a rigorous criterion individual sweeps with either fast or slow mode activity, only single channel patches in which the channel showed (by visual inspection) only (or mainly) one gating mode or clearly separated long periods in the two gating modes were considered. The classification of the activity in a certain patch or period as either slow or fast gating mode was then based on the open/closed time histograms and/or on the voltage dependence of the open probability. In 168 min of recording (from 11 single channel patches), channels containing the ß_1b subunit spent 46% of the time in the slow gating mode. A larger fraction of time (58%, in 183 min of recordings from 12 single channel patches) was spent in the slow gating mode by channels containing the ß_2e subunit. Channels containing the ß_4a subunit spent most of the time in the slow gating mode (68%, in 117 min of recordings from nine single channel patches). The opposite was true for channels containing the ß_3a subunit (34%, in 87 min of recordings from six single channel patches). According to Fisher exact test, the differences in occurrence of the fast and slow gating modes among channels with the different ß subunits are all statistically significant (P < 0.05 or 0.001). Transitions from the slow gating mode (present at the beginning of the recording) to the fast mode were observed in 2, 3, 0, and 2 single channel patches with the ß_1b, ß_2e, ß_3a, and ß_4a subunit, respectively. The opposite transition from the fast gating mode (present at the beginning) to the slow mode was observed only in one single channel patch with the ß_4a subunit. In the remaining single channel patches, the channel remained mainly or only in either the fast (5, 3, 4, and 1 patches) or slow gating mode (4, 6, 2, and 5 patches).

In contrast with the gating properties, the permeation properties of Ca_V2.1 channels were not affected by the type of ß subunit. Ca_V2.1 channels containing the four different ß subunits had identical single channel currents and conductances (Fig. 5).

View larger version (19K): [in this window] [in a new window]   FIGURE 5. The single channel current and conductance of CaV2.1 channels do not depend on the type of ß subunit. Single channel recordings as in Fig. 1 on HEK293 cells stably coexpressing human CaV2.11 (1A-2), 2b-1, and ß1b, ß2e, ß3a, or ß4a subunits. Unitary current, i, as a function of voltage of CaV2.1 channels containing the ß1b (), ß2e (•), ß3a (), and ß4a () subunit. The average i–V relationships were obtained from 5, 6, 4, and 12 patches and the average slope conductance was of 19.5 ± 0.4, 20 ± 0.4, 19 ± 0.9, and 20 ± 0.4 pS for channels containing the ß1b, ß2e, ß3a, and ß4a subunit, respectively.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. The main gating mode of CaV2.1 channels lacking the ß subunit is a low-po mode different from both the fast and the slow gating modes. Single channel recordings as in Fig. 1 on HEK293 cells transiently coexpressing human CaV2.11 (1A-2) and 2b-1 subunits. (A) Consecutive traces at +30 mV from a patch containing a single 1A-2–2b-1 channel. (B) Log–log plots of the open and closed time distributions of the single channel in A. Time constants of the exponential components best fitting the open times: 0.30 and 0.74 ms (relative areas 82 and 18%); time constants for the closed times: 0.69, 11.3, and 34.9 ms (relative areas 31, 36, and 33%). (C) Open probability, po, as a function of voltage for the single channel in A. Fit of the activation curve with a Boltzmann equation gives V1/2 = 44 mV, k = 9.1 mV, po max = 0.46.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Comparison of the gating parameters of the low-po gating mode of CaV2.1 channels lacking the ß subunit and the slow and fast gating modes of CaV2.1 channels containing the ß subunit. Cell attached recordings on HEK293 cells stably coexpressing human 1A-2, 2b-1, and different ß subunits (for the slow and fast gating modes), or transiently expressing 1A-2 and 2b-1 subunits (for the low-po gating mode). (A) Time constants (open and closed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in fast (empty bar), slow (gray bar), and low-po (dark bar) gating modes. For the fast and slow gating modes, the values of CaV2.1 channels containing different ß subunits from 18 (5 with ß1b, 3 with ß2e, 6 with ß3a, and 4 with ß4a) and 13 (5 with ß1b, 4 with ß2e, and 4 with ß4a) single channel patches, respectively, were averaged; for the low-po gating mode, average values were obtained from six patches containing a single 1A-2–2b-1 channel. Statistical significance of difference between the values for the low-po mode and both the slow and fast gating modes, using Student's t test: **, P < 0.001. (B) Average open probability as a function of voltage of single CaV2.1 channels containing different ß subunits in either the fast (open circles, n = 18) or the slow (gray circles, n = 13) gating mode and of single 1A-2–2b-1 channels in the low-po gating mode (triangles, n = 6) as in A. Fit of the activation curves with a Boltzmann equation gives for the low-po mode, V1/2 = 43.6 mV, k = 8.9 mV, po max = 0.40; for the fast mode, V1/2 = 26 mV, k = 5.2 mV, po max = 0.45; and for the slow mode, V1/2 = 33 mV, k = 4.2 mV, po max = 0.53. The different values of po max for the slow and fast gating modes (in contrast with the similar values in Fig. 1 C) are mainly due to different fractions of time spent by the channels in the b mode (Fellin et al., 2004). (C) Average cumulative first latency histograms of single 1A-2–2b-1 channels in the low-po gating mode (from n = 6 single channel patches) and of single 1A-2–2b–ß1b channels in either the fast or the slow gating mode (from n = 5 and 5 single channel patches, respectively). To obtain the histograms, only sweeps with activity were considered. See text for the parameters of the exponential components best fitting the histograms.

In addition to the prevailing low-p_o mode of gating, Ca_V2.1 channels containing only _1A2 and _2b-1 subunits showed a high-p_o mode of gating similar to the fast gating mode of the _1A2–_2b1-ß channels. In three single channel patches, the average open probability at +30 mV of the channel in this gating mode was 0.267 ± 0.04; the open time constants were 0.58 ± 0.22 and 1.4 ± 0.2 ms with relative contribution of 56 ± 4 and 44% and the closed time constants were 0.45 ± 0.05, 2.4 ± 0.1, and 8.8 ± 1.6 ms with relative contributions of 54 ± 4, 30 ± 2, and 16 ± 2%. The half voltage of activation was 27 ± 2 mV (with k = 4.9 ± 0.2). Much less frequently, _1A2–_2b-1 channels also showed a mode of gating similar to the slow mode. One may wonder whether the fast and slow gating modes recorded in cells transfected with only _1A2 and _2b-1 subunits may correspond to channels containing endogenous ß subunits (that might be expressed at such low level to be undetectable by Western and Northern blots; Meir et al., 2000). This interpretation appears unlikely in light of the following observation. A low-p_o mode of gating very similar to that just described was the prevailing gating mode present (together with the fast and the rarer slow gating mode) in our initial single channel recordings from the cell line PB1-14, which was supposed to stably express _1A2, _2b, and ß_3a subunits. The low-p_o mode of gating was however totally absent after transfection of PB1-14 cells with ß_3a cDNA, suggesting that for some reasons these cells were not expressing sufficient amounts of ß_3a subunits, and therefore most of the recorded single channels lacked the ß_3a subunit. Indeed, the single channel open probability (0.07 ± 0.01, n = 3), the open and closed time constants (_o1 = 0.26 ± 0.04 ms, _o2 = 0.97 ± 0.26 ms; _c1 = 0.72 ± 0.10 ms, _c2 = 8.8 ± 0.3 ms, _c3 = 34 ± 5 ms, n = 3), and the fraction of nulls (64 ± 6%, n = 4) obtained for the low-p_o gating pattern in PB1-14 cells were very similar to those obtained for the low-p_o activity in HEK293 cells transfected with only _1A2 and _2b-1 subunits. Interestingly, in two single channel patches from PB1-14 cells, transitions of the same channel from the slow to the low-p_o gating mode, and in one of them, from the low-p_o to the fast gating mode and back to the low-p_o mode were observed. This observation is consistent with the hypothesis that the three gating patterns reflect different modes of gating of a Ca_V2.1 channel lacking a ß subunit rather than different combinations of Ca_V2.1₁ subunits and auxiliary subunits. The low-p_o gating mode shown in Figs. 6 and 7 is the prevailing mode of gating of Ca_V2.1 channels without a ß subunit, but is completely absent in channels containing a ß subunit. In fact, in hundreds of cell-attached patches on HEK293 cells transiently transfected with human Ca_V2.1₁, _2b-1, and ß_2e subunits, the low-p_o gating mode was never observed (Hans et al., 1999; Tottene et al., 2002). Likewise, it was never observed in cells transiently transfected with only _1A2 and ß_2e subunits (n = 8).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The main findings reported in this paper can be summarized as follows. (a) Single recombinant human Ca_V2.1 channels show two different modes of gating (slow and fast), characterized by different mean closed times and latency to first opening (both longer when the channel is in the slow mode), different voltage dependence of the open probability (larger depolarizations are necessary to activate the channel in the slow mode), different kinetics of inactivation (slower in the slow mode), and different voltage dependence of steady-state inactivation (occurring at less negative voltages when the channel is in the slow mode). (b) Ca_V2.1 channels containing any of the ß subtypes (ß_1b, ß_2e, ß_3a, or ß_4a) can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. With all ß subunits, inactivation is more rapid and steady-state inactivation occurs at more negative voltages in the fast than in the slow gating mode. However, in both gating modes, Ca_V2.1 channels display different rates of inactivation and different steady-state inactivation depending on the ß subtype, with ß_2e giving a considerably slower rate of inactivation and less negative voltage range of steady-state inactivation than the other ß subunits. (c) The relative occurrence of the slow and fast gating modes of Ca_V2.1 channels is modulated by the type of auxiliary ß subunit; ß_3a promotes the fast mode, whereas ß_4a promotes the slow mode of recombinant Ca_V2.1 channels expressed in HEK293 cells. (d) The prevailing mode of gating of Ca_V2.1 channels lacking a ß subunit is a low-p_o mode different from both the fast and the slow gating modes. A channel in the low-p_o mode shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages, displaying a shallower voltage dependence, than in either the fast or the slow gating mode.

Modal gating appears to be a highly conserved feature among different ion channel types. Discrete modes of single channel activity, presumably corresponding to different sets of protein conformations, have been described for voltage-dependent Ca²⁺ (Hess and Tsien, 1984; Yue et al., 1990; Plummer and Hess, 1991; Delcour et al., 1993; Forti and Pietrobon, 1993; Rittenhouse and Hess, 1994; Mantegazza et al., 1995), Na⁺ (Patlak and Ortiz, 1986; Nilius, 1988; Moorman et al., 1990; Zhou et al., 1991; Alzheimer et al., 1993; Bohle et al., 1998), K⁺ (Marrion, 1996; Singer-Lahat et al., 1999), and Cl^– (Blatz and Magleby, 1986) channels, and also for Ca²⁺-activated and G protein–activated K⁺ channels (McManus and Magleby, 1988; Smith and Ashford, 1998; Yakubovich et al., 2000) and for ligand-activated channels (for example see Naranjo and Brehm, 1993; Popescu and Auerbach, 2003). In most cases, it remains unclear whether the transitions between modes of gating (occurring in time frames ranging from hundreds of milliseconds to several minutes) reflect slow conformational changes intrinsic to the channels or conformational changes driven by chemical reactions or interaction with other proteins. However, there are several clear instances in which phosphorylation–dephosphorylation reactions either modulate the rate constants of interconversion between different gating modes of the channel or drive the channel into different gating modes corresponding to different states of phosphorylation (c.f. for L-type Ca²⁺ channels, Ochi and Kawashima, 1990; Yue et al., 1990; Herzig et al., 1993; Ono and Fozzard, 1993; Dzhura et al., 2000; for different K⁺ channels, Marrion, 1996; Smith and Ashford, 1998; Singer-Lahat et al., 1999; for a cation channel in Aplysia neurons, Wilson and Kaczmarek, 1993). In other cases, it has been shown that the interconversion between different gating modes is either modulated or driven by the interaction of the channel with other proteins, e.g. auxiliary subunits (Naranjo and Brehm, 1993; Chang et al., 1996; Singer-Lahat et al., 1999; Wakamori et al., 1999; Meir et al., 2000) and/or G protein subunits (Delcour et al., 1993; Lee and Elmslie, 2000; Colecraft et al., 2001), or calmodulin (Imredy and Yue, 1994; Peterson et al., 1999; Zuhlke et al., 1999). In addition, voltage dependence of the rate constants of interconversion between different gating modes has been clearly shown for L-type Ca²⁺ channels (Pietrobon and Hess, 1990; Forti and Pietrobon, 1993; Cloues et al., 1997; Hivert et al., 1999).

This (and the following, Fellin et al., 2004) is the first report of modal gating of Ca_V2.1 channels. The fast and slow modes of gating, described in this study, are quite different from the gating modes previously reported for L-type and N-type Ca²⁺ channels (Hess and Tsien, 1984; Yue et al., 1990; Plummer and Hess, 1991; Delcour et al., 1993; Rittenhouse and Hess, 1994; Mantegazza et al., 1995). The distinguishing features are (a) the long mean lifetimes (time frame of several minutes), pointing to quite slow rate constants for the transitions between the two gating modes, and (b) the different kinetics and voltage dependence of inactivation of the channel in the two gating modes, together with different closed times (but similar open times) and different voltage dependence of activation. The gating modes of L-type Ca²⁺ channels are characterized by large differences in open and closed times of the channel in the different modes, leading to very different open probabilities, without reported clear differences in inactivation properties (Hess and Tsien, 1984; Yue et al., 1990; Mantegazza et al., 1995). Gating modes with different kinetics and voltage dependence of inactivation have been reported for N-type Ca²⁺ channels (Plummer and Hess, 1991), but the much shorter mean lifetime (few seconds) of the inactivating and noninactivating gating modes of N-type channels and the similar open and closed times of the channel in the two gating modes clearly distinguish the modal behavior of N-type (Ca_V2.2) channels from that of Ca_V2.1 channels described here (but see Fellin et al., 2004). On the other hand, inactivating and noninactivating gating modes with mean lifetimes in the time frame of several minutes, which share several properties with the fast and slow gating modes of Ca_V2.1 channels, have been described for Na⁺ and K_V1.1 channels (Zhou et al., 1991; Singer-Lahat et al., 1999; Tabarean et al., 1999). For both Na⁺ and K⁺ channels, there are indirect evidences, from macroscopic current recordings, that interaction of the channel with the cytoskeleton and with Gß subunits either modulate or drive switching between the inactivating and noninactivating gating modes (Levin et al., 1996; Ma et al., 1997; Jing et al., 1999; Tabarean et al., 1999). It will be interesting to investigate with future work whether similar protein interactions modulate or drive switching between the fast and slow gating modes of Ca_V2.1 channels. For the time being, we have established that interaction with syntaxin1A is not involved in this mode switching, since incubation with botulinum toxin C1, to cleave syntaxin, did not alter modal gating of human Ca_V2.1 channels expressed in HEK293 cells (unpublished data; c.f. Sutton et al., 1999).

Our data suggest that the equilibrium between the slow and fast gating modes of Ca_V2.1 channels is modulated by the type of auxiliary ß subunit. Thus, ß subunits exert a dual regulation of inactivation of Ca_V2.1 channels; on one hand, the ß subtype affects the inactivation properties of the channel in each gating mode, and, on the other hand, it affects the fraction of time spent by the channel in the slowly inactivating and more rapidly inactivating gating modes. The order of efficiency with which the different ß subunits shift the equilibrium between gating modes toward the inactivating fast mode (ß_3a > ß_1b> ß_2e > ß_4a) is quite different from that with which they increase the rate of inactivation of the channel (ß_4a > ß_1b ß_3a >> ß_2e, judging from the fraction of current inactivating during 720 ms, or ß_3a ß_1b > ß_4a >> ß_2e, judging from the time constant of inactivation of the ensemble current of the inactivating traces) or shift steady-state inactivation toward negative voltages (ß_4a ß_1b > ß_3a >> ß_2e). These observations suggest that the ß subunit modulation of inactivation properties and of modal gating occurs by different mechanisms and molecular interactions. It might be interesting to note that the two ß subunits that seem to favor the slow gating mode are those that specifically interact with the COOH and NH₂ terminals of the Ca_V2.1₁ subunit (Walker et al., 1998, 1999), with ß_4a showing both a higher affinity for these binding sites and a larger occurrence of the slow gating mode.

The dual independent regulation of channel inactivation by ß subunits might contribute to explain the disagreement in the literature regarding the order of efficiencies with which different ß subunits modulate the inactivation properties of Ca_V2.1 channels (Stea et al., 1994; De Waard and Campbell, 1995; Restituito et al., 2000). In fact, different splice variants of Ca_V2.1₁ might well display a different equilibrium between the slow and fast gating modes or a different regulation of modal gating by ß subunits. Moreover, as found for many channels, mode switching might be modulated by other factors (e.g., interactions with other proteins or metabolic processes) that may be cell type specific.

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