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Journal of Experimental Biology, Vol 202, Issue 4 429-440, Copyright © 1999 by Company of Biologists
JOURNAL ARTICLES |
J GarcIA-Colunga, R Valdiosera and U GarcIA
Center of Neurobiology, National University of Mexico, Campus Juriquilla, Queretaro, Biophysics and Neuroscience, CINVESTAV, 07000 Mexico. ugarcia@fisio.cinvestav.mx
Inward Ca2+ current through voltage-gated Ca2+ channels was recorded from freshly dissociated crayfish X-organ (XO) neurones using the whole-cell voltage-clamp technique. Changing the holding potential from -50 to -90 mV had little effect on the characteristics of the current-voltage relationship: neither the time course nor the amplitude of the Ca2+ current was affected. Inactivation of the Ca2+ current was observed over a small voltage range, between -35 and -10 mV, with half-inactivation at -20 mV. The activation of the Ca2+ current was modelled using Hodgkin-Huxley kinetics. The time constant of activation, &tgr; m, was 568+/-66 micros at -20 mV and decreased gradually to 171+/-23 micros at 40 mV (means +/- s.e.m., N=5). The steady-state activation, m(infinity), was fitted with a Boltzmann function, with a half-activation voltage of -7.45 mV and an apparent threshold at -40 mV. The instantaneous current-voltage relationship was adjusted using the Goldman-Hodgkin-Katz constant-field equation, giving a permeation of 4.95x10(-5 )cm s-1. The inactivation of the Ca2+ current in XO neurones was dependent on previous entry of Ca2+. Using a double-pulse protocol, the inactivation was fitted to a U-shaped curve with a maximal inactivation of 35 % at 30 mV. The time course of the recovery from inactivation was fitted with an exponential function. The time constants were 17+/-2.6 ms for a prepulse of 10 ms and 31+/-3.2 ms for a prepulse of 20 ms. The permeability sequence of the Ca2+ channels was as follows: Ba2+>Sr2+~Ca2+>>Mg2+. Other divalent cations blocked the Ca2+ current, and their effects were voltage-dependent; the potency of blockage was Cd2+~Zn2+>>Co2+~Ni2+. The peptide &ohgr; -agatoxin-IVA, a selective toxin for P-type Ca2+ channels, blocked 85 % of the Ca2+ current in XO neurones at 200 nmol l-1, but the current was insensitive to dihydropyridines, phenylalkylamines, &ohgr; -conotoxin-GVIA and &ohgr; -conotoxin-MVIIC, which are blockers of L-, N- and Q-type Ca2+ channels, respectively. From the voltage- and Ca2+-dependent kinetics, the higher permeability to Ba2+ than to Ca2+ and the higher sensitivity of the current to Cd2+ than to Ni2+, we conclude that the Ca2+ current in XO neurones is generated by high-voltage-activated (HVA) channels. Furthermore, its blockage by &ohgr; -agatoxin-IVA suggests that it is mainly generated through P-type Ca2+ channels.
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