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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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Switching between the b and nb gating modes produces reversible uncoupling of inactivation of CaV2.1 channels. Single channel recordings and voltage protocol as in Fig. 4. Representative current traces from a patch containing a single CaV2.1 channel in the fast gating mode showing frequent switching between the nb (left traces) and b (right traces) gating modes, as inferred from the gating pattern in the short pulse at +50 mV following the prepulse at +30 mV. Below the traces, the corresponding ensemble average currents at +30 mV for the nb (left, n = 83) and b (right, n = 44) gating modes are shown. Separate average currents for the b and nb gating modes at +30 mV were obtained considering the inactivating traces within a string of noninactivating sweeps showing b or nb mode activity as b or nb traces, respectively, and those at the beginning or at the end of an nb period as nb traces. The bottom panel shows the channel activity in consecutive sweeps, classified as b, nb, , and inactivating. Note that channel inactivation at +30 mV occurs almost exclusively during periods with nb mode activity. The same is true for s; the probability that an nb sweep was preceded or followed by a  trace was 39/133 pairs = 0.28, whereas the probability that a b trace was preceded or followed by a  trace was 1/73 pairs = 0.014 (in this calculation, inactivating traces that were within and at the beginning or end of a string in nb mode were considered as nb sweeps).
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fig7: Switching between the b and nb gating modes produces reversible uncoupling of inactivation of CaV2.1 channels. Single channel recordings and voltage protocol as in Fig. 4. Representative current traces from a patch containing a single CaV2.1 channel in the fast gating mode showing frequent switching between the nb (left traces) and b (right traces) gating modes, as inferred from the gating pattern in the short pulse at +50 mV following the prepulse at +30 mV. Below the traces, the corresponding ensemble average currents at +30 mV for the nb (left, n = 83) and b (right, n = 44) gating modes are shown. Separate average currents for the b and nb gating modes at +30 mV were obtained considering the inactivating traces within a string of noninactivating sweeps showing b or nb mode activity as b or nb traces, respectively, and those at the beginning or at the end of an nb period as nb traces. The bottom panel shows the channel activity in consecutive sweeps, classified as b, nb, , and inactivating. Note that channel inactivation at +30 mV occurs almost exclusively during periods with nb mode activity. The same is true for s; the probability that an nb sweep was preceded or followed by a trace was 39/133 pairs = 0.28, whereas the probability that a b trace was preceded or followed by a trace was 1/73 pairs = 0.014 (in this calculation, inactivating traces that were within and at the beginning or end of a string in nb mode were considered as nb sweeps).

Mentions: Temporal correlation among sweeps with low-po (b) and high-po (nb) gating patterns and mean lifetime of the b and nb modes. Cell-attached patch-clamp recordings as in Fig. 1 on HEK293 cells stably or transiently coexpressing human α1A-2, β1b, and α2bδ-1 subunits. (A, a and b) Sequences of sweeps in two representative single channel patches, classified as b or nb on the basis of a cut-off po value of 0.45 at +50 mV. N represents sweeps without activity (s). (B, a and b) Number of pairs of consecutive traces in the four different combinations (b-b, b-nb, nb-b, and nb-nb) in the two single channel patches shown in A, a and b. Pairs containing s were not counted. (C) Number of consecutive traces with either b or nb gating mode activity in 13 single channel patches, plotted as histograms with binwidth of one trace. Fitting with a single exponential function gives mean lifetimes of 1.6 traces (6.4 s) for the nb gating mode and 1.8 traces (7.2 s) for the b gating mode. If s within a string of sweeps with nb or b mode activity were considered as part of the gating mode, then the mean lifetime of the nb gating mode would increase relative to that of the b mode (compare Fig. 7).


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)

Switching between the b and nb gating modes produces reversible uncoupling of inactivation of CaV2.1 channels. Single channel recordings and voltage protocol as in Fig. 4. Representative current traces from a patch containing a single CaV2.1 channel in the fast gating mode showing frequent switching between the nb (left traces) and b (right traces) gating modes, as inferred from the gating pattern in the short pulse at +50 mV following the prepulse at +30 mV. Below the traces, the corresponding ensemble average currents at +30 mV for the nb (left, n = 83) and b (right, n = 44) gating modes are shown. Separate average currents for the b and nb gating modes at +30 mV were obtained considering the inactivating traces within a string of noninactivating sweeps showing b or nb mode activity as b or nb traces, respectively, and those at the beginning or at the end of an nb period as nb traces. The bottom panel shows the channel activity in consecutive sweeps, classified as b, nb, , and inactivating. Note that channel inactivation at +30 mV occurs almost exclusively during periods with nb mode activity. The same is true for s; the probability that an nb sweep was preceded or followed by a  trace was 39/133 pairs = 0.28, whereas the probability that a b trace was preceded or followed by a  trace was 1/73 pairs = 0.014 (in this calculation, inactivating traces that were within and at the beginning or end of a string in nb mode were considered as nb sweeps).
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getmorefigures.php?uid=PMC2234006&req=5

fig7: Switching between the b and nb gating modes produces reversible uncoupling of inactivation of CaV2.1 channels. Single channel recordings and voltage protocol as in Fig. 4. Representative current traces from a patch containing a single CaV2.1 channel in the fast gating mode showing frequent switching between the nb (left traces) and b (right traces) gating modes, as inferred from the gating pattern in the short pulse at +50 mV following the prepulse at +30 mV. Below the traces, the corresponding ensemble average currents at +30 mV for the nb (left, n = 83) and b (right, n = 44) gating modes are shown. Separate average currents for the b and nb gating modes at +30 mV were obtained considering the inactivating traces within a string of noninactivating sweeps showing b or nb mode activity as b or nb traces, respectively, and those at the beginning or at the end of an nb period as nb traces. The bottom panel shows the channel activity in consecutive sweeps, classified as b, nb, , and inactivating. Note that channel inactivation at +30 mV occurs almost exclusively during periods with nb mode activity. The same is true for s; the probability that an nb sweep was preceded or followed by a trace was 39/133 pairs = 0.28, whereas the probability that a b trace was preceded or followed by a trace was 1/73 pairs = 0.014 (in this calculation, inactivating traces that were within and at the beginning or end of a string in nb mode were considered as nb sweeps).
Mentions: Temporal correlation among sweeps with low-po (b) and high-po (nb) gating patterns and mean lifetime of the b and nb modes. Cell-attached patch-clamp recordings as in Fig. 1 on HEK293 cells stably or transiently coexpressing human α1A-2, β1b, and α2bδ-1 subunits. (A, a and b) Sequences of sweeps in two representative single channel patches, classified as b or nb on the basis of a cut-off po value of 0.45 at +50 mV. N represents sweeps without activity (s). (B, a and b) Number of pairs of consecutive traces in the four different combinations (b-b, b-nb, nb-b, and nb-nb) in the two single channel patches shown in A, a and b. Pairs containing s were not counted. (C) Number of consecutive traces with either b or nb gating mode activity in 13 single channel patches, plotted as histograms with binwidth of one trace. Fitting with a single exponential function gives mean lifetimes of 1.6 traces (6.4 s) for the nb gating mode and 1.8 traces (7.2 s) for the b gating mode. If s within a string of sweeps with nb or b mode activity were considered as part of the gating mode, then the mean lifetime of the nb gating mode would increase relative to that of the b mode (compare Fig. 7).

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