Mode and Reversible Uncoupling of Inactivation

The single channel gating properties of human Ca V 2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human Ca V 2.1 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 Ca V 2.1 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 Ca V 2.1 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 Ca V 2.1 channels. In fact, a Ca V 2.1 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 Ca V 2.1 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 Ca V 2.1 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 Ca 2 -dependent processes. key words: Ca 2 channel • gating mode • synaptic transmission • familial hemiplegic migraine • channelopathy I N T R O D U C T I O N Ca 2 influx through Ca V 2.1 (P/Q-type) calcium channels plays a central role in controlling neurotransmitter release in the brain (Dunlap et al., 1995; Mintz et al., 1995; Wu et al., 1999; Qian and Noebels, 2001). Ca V 2.1 channels are also involved in regulating neural and cortical network excitability, synaptic integration, and gene expression (Bayliss et al., 1997; Magee et al., 1998; Pineda et al., 1998; Sutton et al., 1999; Mori et al., 2000; van den Maagdenberg et al., 2004). The time course and amplitude of Ca 2 influx through Ca V channels in response to physiological electrical activity is determined by their single channel gating and permeation properties. These properties are then crucial in determining the time course and magnitude of the various Ca 2 -dependent processes controlled by Ca V 2.1 channels. The single channel study reported in the preceding paper (Luvisetto et al., 2004) revealed a complex modal gating of human Ca V 2.1 channels; single channels can display two modes of gating characterized by different latencies to first opening and different mean closed times, different voltage dependence of the open probability, different kinetics of inactivation, and different voltage dependence of steady-state inactivation. Different latencies to first opening and different open probabilities can alter the timing and the magnitude of action potential–evoked Ca 2 influx , and can profoundly affect both the timing and magnitude of neurotransmission controlled by Ca V 2.1 channels (Borst and Sakmann, 1998; Sabatini and Regehr, 1999). Different properties of inactivation can substantially alter Ca 2 influx in response to repetitive firing waveforms (Liu et al., 2003). Most of the single channel recordings in Luvisetto et al. (2004) were done at voltages 30 mV lower than those attained at the peak of action potentials recorded in neurons (taking into account the difference in surface potential due to the high divalent ion concentration used in single channel recordings to allow resolution of single channel gating events; c.f. Tottene et al., 2002). Therefore, it seemed interesting to examine the gating properties of the channels at higher voltages. This study revealed frequent switching of 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@unipd.it Abbreviation used in this paper: HEK, human embryonic kidney. on A uust 0, 2017 jgp.rress. org D ow nladed fom