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Modal gating of human CaV2.1 (P/Q-type) calcium channels: II. the b mode and reversible uncoupling of inactivation.

Fellin T, Luvisetto S, Spagnolo M, Pietrobon D - J. Gen. Physiol. (2004)

Bottom Line: Physiol. 124:445-461).In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly.Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biomedical Sciences, University of Padova, Viale G. Colombo, 3 35121 Padova, Italy.

ABSTRACT
The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445-461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.

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At V < +40 mV, a CaV2.1 channel in the b gating mode has similar open probability and open and closed time distributions than in the nb mode. Cell-attached patch-clamp recordings from single channel patches as in Fig. 3, but with a voltage protocol in which 540-ms-long depolarizations to either 20 or 30 mV were followed by 180-ms-long depolarizations to +50 mV. (A) Representative single channel current traces from a patch containing a single CaV2.1 channel, which alternates between the nb and b modes of gating, are shown together with log–log plots of the open and closed time distributions at +30 mV, obtained for the nb (left) and the b gating mode (right), after separation of the traces in the two gating modes on the basis of the activity in the short pulse at +50 mV that follows the depolarization at +30 mV. Judging from the activity at +30 mV, the single channel in the patch was in the fast gating mode for the entire period, and, at any given voltage, had a higher open probability than usually found for this gating mode (e.g., the maximal po at +50 mV in the nb gating mode was 0.86, to be compared with values of 0.6–0.7 for most of the single channel analyzed), mainly due to unusually long mean open times (Luvisetto et al., 2004). The dark solid line in each plot is the best-fitting sum of exponential components (two for the open times and three for the closed times), each shown as a dotted line (minimum number of components indicated by the maximum likelihood ratio test); time constants for the open times, 1.04 and 2.06 ms (relative areas 41 and 59%) for the b gating mode, and 1.56 and 3.42 ms (relative areas 48 and 52%) for the nb gating mode; time constants for the closed times, 0.31, 1.56, and 3.84 ms (relative areas 50, 42, and 8%) for the b gating mode, and 0.32, 1.29, and 3.7 ms (relative areas 70, 20, and 10%) for the nb mode. Thanks to the unusually long mean open times, this channel showed a small but significant difference in the time constants best fitting the open time distributions for the b and nb gating modes, in contrast with the similar average values shown in B. (B) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the fast-nb (empty bar) and the fast-b (gray bar) gating modes. Average values were obtained from four patches containing a single channel in the fast gating mode. (C) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the slow-nb (empty bar) and the slow-b (gray bar) gating modes. Average values from three single channel patches.
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fig4: At V < +40 mV, a CaV2.1 channel in the b gating mode has similar open probability and open and closed time distributions than in the nb mode. Cell-attached patch-clamp recordings from single channel patches as in Fig. 3, but with a voltage protocol in which 540-ms-long depolarizations to either 20 or 30 mV were followed by 180-ms-long depolarizations to +50 mV. (A) Representative single channel current traces from a patch containing a single CaV2.1 channel, which alternates between the nb and b modes of gating, are shown together with log–log plots of the open and closed time distributions at +30 mV, obtained for the nb (left) and the b gating mode (right), after separation of the traces in the two gating modes on the basis of the activity in the short pulse at +50 mV that follows the depolarization at +30 mV. Judging from the activity at +30 mV, the single channel in the patch was in the fast gating mode for the entire period, and, at any given voltage, had a higher open probability than usually found for this gating mode (e.g., the maximal po at +50 mV in the nb gating mode was 0.86, to be compared with values of 0.6–0.7 for most of the single channel analyzed), mainly due to unusually long mean open times (Luvisetto et al., 2004). The dark solid line in each plot is the best-fitting sum of exponential components (two for the open times and three for the closed times), each shown as a dotted line (minimum number of components indicated by the maximum likelihood ratio test); time constants for the open times, 1.04 and 2.06 ms (relative areas 41 and 59%) for the b gating mode, and 1.56 and 3.42 ms (relative areas 48 and 52%) for the nb gating mode; time constants for the closed times, 0.31, 1.56, and 3.84 ms (relative areas 50, 42, and 8%) for the b gating mode, and 0.32, 1.29, and 3.7 ms (relative areas 70, 20, and 10%) for the nb mode. Thanks to the unusually long mean open times, this channel showed a small but significant difference in the time constants best fitting the open time distributions for the b and nb gating modes, in contrast with the similar average values shown in B. (B) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the fast-nb (empty bar) and the fast-b (gray bar) gating modes. Average values were obtained from four patches containing a single channel in the fast gating mode. (C) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the slow-nb (empty bar) and the slow-b (gray bar) gating modes. Average values from three single channel patches.

Mentions: In the recordings at +30 mV, there was no evidence of the b gating mode clearly seen at higher voltages (Luvisetto et al., 2004; and c.f. single peak in po histograms in Fig. 2). To try to understand this finding, we adopted the double pulse protocol shown in Fig. 4, in which a prepulse to +20 or +30 mV was followed by a short pulse to +50 mV. We could then separate (using the cut-off po value of 0.45) the traces in which the channel was in the b gating mode during the short pulse at +50 mV (and presumably also during the prepulse at +30 or +20 mV) from those in which it was in the nb gating mode. As shown by the representative traces in Fig. 4, the single channel activity at +30 mV (and even more at +20 mV) was similar in the b and nb gating modes (compare first two and last two traces at both voltages). Also similar were the open as well as the closed time histograms of the channel in the two gating modes at +30 mV. Indeed, Fig. 4 B shows that the average values of the open and closed time constants of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the fast-nb or the fast-b gating mode were not significantly different. The open probability at +30 mV was also similar in b (0.36 ± 0.03) and nb (0.38 ± 0.05) gating modes. Similar open probabilities (po = 0.2 ± 0.02 and 0.21 ± 0.04 in the b and nb gating modes, respectively) and similar open and closed time histograms (Fig. 4 C) were obtained also for single channels in the slow-b and slow-nb gating modes. The single peaks at +30 and +20 mV in the po histograms of Fig. 2 and the lack of evidence for the b gating mode at +30 mV are then due to the fact that, at V < +40 mV, the open and closed time distributions of a channel in the b and nb gating modes are so similar that the two gating modes become indistinguishable.


Modal gating of human CaV2.1 (P/Q-type) calcium channels: II. the b mode and reversible uncoupling of inactivation.

Fellin T, Luvisetto S, Spagnolo M, Pietrobon D - J. Gen. Physiol. (2004)

At V < +40 mV, a CaV2.1 channel in the b gating mode has similar open probability and open and closed time distributions than in the nb mode. Cell-attached patch-clamp recordings from single channel patches as in Fig. 3, but with a voltage protocol in which 540-ms-long depolarizations to either 20 or 30 mV were followed by 180-ms-long depolarizations to +50 mV. (A) Representative single channel current traces from a patch containing a single CaV2.1 channel, which alternates between the nb and b modes of gating, are shown together with log–log plots of the open and closed time distributions at +30 mV, obtained for the nb (left) and the b gating mode (right), after separation of the traces in the two gating modes on the basis of the activity in the short pulse at +50 mV that follows the depolarization at +30 mV. Judging from the activity at +30 mV, the single channel in the patch was in the fast gating mode for the entire period, and, at any given voltage, had a higher open probability than usually found for this gating mode (e.g., the maximal po at +50 mV in the nb gating mode was 0.86, to be compared with values of 0.6–0.7 for most of the single channel analyzed), mainly due to unusually long mean open times (Luvisetto et al., 2004). The dark solid line in each plot is the best-fitting sum of exponential components (two for the open times and three for the closed times), each shown as a dotted line (minimum number of components indicated by the maximum likelihood ratio test); time constants for the open times, 1.04 and 2.06 ms (relative areas 41 and 59%) for the b gating mode, and 1.56 and 3.42 ms (relative areas 48 and 52%) for the nb gating mode; time constants for the closed times, 0.31, 1.56, and 3.84 ms (relative areas 50, 42, and 8%) for the b gating mode, and 0.32, 1.29, and 3.7 ms (relative areas 70, 20, and 10%) for the nb mode. Thanks to the unusually long mean open times, this channel showed a small but significant difference in the time constants best fitting the open time distributions for the b and nb gating modes, in contrast with the similar average values shown in B. (B) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the fast-nb (empty bar) and the fast-b (gray bar) gating modes. Average values were obtained from four patches containing a single channel in the fast gating mode. (C) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the slow-nb (empty bar) and the slow-b (gray bar) gating modes. Average values from three single channel patches.
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fig4: At V < +40 mV, a CaV2.1 channel in the b gating mode has similar open probability and open and closed time distributions than in the nb mode. Cell-attached patch-clamp recordings from single channel patches as in Fig. 3, but with a voltage protocol in which 540-ms-long depolarizations to either 20 or 30 mV were followed by 180-ms-long depolarizations to +50 mV. (A) Representative single channel current traces from a patch containing a single CaV2.1 channel, which alternates between the nb and b modes of gating, are shown together with log–log plots of the open and closed time distributions at +30 mV, obtained for the nb (left) and the b gating mode (right), after separation of the traces in the two gating modes on the basis of the activity in the short pulse at +50 mV that follows the depolarization at +30 mV. Judging from the activity at +30 mV, the single channel in the patch was in the fast gating mode for the entire period, and, at any given voltage, had a higher open probability than usually found for this gating mode (e.g., the maximal po at +50 mV in the nb gating mode was 0.86, to be compared with values of 0.6–0.7 for most of the single channel analyzed), mainly due to unusually long mean open times (Luvisetto et al., 2004). The dark solid line in each plot is the best-fitting sum of exponential components (two for the open times and three for the closed times), each shown as a dotted line (minimum number of components indicated by the maximum likelihood ratio test); time constants for the open times, 1.04 and 2.06 ms (relative areas 41 and 59%) for the b gating mode, and 1.56 and 3.42 ms (relative areas 48 and 52%) for the nb gating mode; time constants for the closed times, 0.31, 1.56, and 3.84 ms (relative areas 50, 42, and 8%) for the b gating mode, and 0.32, 1.29, and 3.7 ms (relative areas 70, 20, and 10%) for the nb mode. Thanks to the unusually long mean open times, this channel showed a small but significant difference in the time constants best fitting the open time distributions for the b and nb gating modes, in contrast with the similar average values shown in B. (B) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the fast-nb (empty bar) and the fast-b (gray bar) gating modes. Average values were obtained from four patches containing a single channel in the fast gating mode. (C) Time constants (τopen and τclosed) and relative areas (%) of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the slow-nb (empty bar) and the slow-b (gray bar) gating modes. Average values from three single channel patches.
Mentions: In the recordings at +30 mV, there was no evidence of the b gating mode clearly seen at higher voltages (Luvisetto et al., 2004; and c.f. single peak in po histograms in Fig. 2). To try to understand this finding, we adopted the double pulse protocol shown in Fig. 4, in which a prepulse to +20 or +30 mV was followed by a short pulse to +50 mV. We could then separate (using the cut-off po value of 0.45) the traces in which the channel was in the b gating mode during the short pulse at +50 mV (and presumably also during the prepulse at +30 or +20 mV) from those in which it was in the nb gating mode. As shown by the representative traces in Fig. 4, the single channel activity at +30 mV (and even more at +20 mV) was similar in the b and nb gating modes (compare first two and last two traces at both voltages). Also similar were the open as well as the closed time histograms of the channel in the two gating modes at +30 mV. Indeed, Fig. 4 B shows that the average values of the open and closed time constants of the exponential components best fitting the open and closed time distributions at +30 mV of single CaV2.1 channels in the fast-nb or the fast-b gating mode were not significantly different. The open probability at +30 mV was also similar in b (0.36 ± 0.03) and nb (0.38 ± 0.05) gating modes. Similar open probabilities (po = 0.2 ± 0.02 and 0.21 ± 0.04 in the b and nb gating modes, respectively) and similar open and closed time histograms (Fig. 4 C) were obtained also for single channels in the slow-b and slow-nb gating modes. The single peaks at +30 and +20 mV in the po histograms of Fig. 2 and the lack of evidence for the b gating mode at +30 mV are then due to the fact that, at V < +40 mV, the open and closed time distributions of a channel in the b and nb gating modes are so similar that the two gating modes become indistinguishable.

Bottom Line: Physiol. 124:445-461).In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly.Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biomedical Sciences, University of Padova, Viale G. Colombo, 3 35121 Padova, Italy.

ABSTRACT
The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445-461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.

Show MeSH
Related in: MedlinePlus