P-type Ca2+ current in crayfish peptidergic neurones

Inward Ca2+ current through voltage-gated Ca2+ channels was recorded from f reshly dissociated crayfish X-organ (XO) neurones using the whole-cell volt age-clamp technique, Changing the holding potential from -50 to -90 mV had little effect on the characteristics of the current-voltage relationship: n either the time course nor the amplitude of the Ca2+ current was affected. Inactivation of the Ca2+ current was observed over a small voltage range, b etween -35 and -10 mV, with half-inactivation at -20 mV, The activation of the Ca2+ current was modelled using Hodgkin-Huxley kinetics. The time const ant of activation, tau(m), was 568+/-66 mu s at -20 mV and decreased gradua lly to 171+/-23 mu s at 40 mV (means +/- S.E.M., N=5). The steady-state act ivation, m(infinity), was fitted with a Boltzmann function, with a half-act ivation voltage of -7.45 mV and an apparent threshold at -40 mV. The instan taneous 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 previ ous entry of Ca2+, Using a double-pulse protocol, the inactivation was fitt ed to a U-shaped curve with a maximal inactivation of 35 % at 30 mV, The ti me 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 3 1+/-3.2 ms for a prepulse of 20 ms. The permeability sequence of the Ca2+ c hannels was as follows: Ba2+>Sr(2+)approximate to Ca(2+)much greater than M g2+. Other divalent cations blocked the Ca2+ current, and their effects wer e voltage-dependent; the potency of blockage was Cd(2+)approximate to Zn(2)much greater than Co(2+)approximate to Ni2+. The peptide omega-agatoxin-IV A, a selective toxin for P-type Ca2+ channels, blocked 85 % of the Ca2+ cur rent in XO neurones at 200 nmol l(-1), but the current was insensitive to d ihydropyridines, phenylalkylamines, omega-conotoxin-GVIA and omega-conotoxi n-MVIIC, which are blockers of L-, N- and Q-type Ca2+ channels, respectivel y, From the voltage- and Ca2+-dependent kinetics, the higher permeability t o 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 h igh-voltage-activated (HVA) channels. Furthermore, its blockage by omega-ag atoxin-IVA suggests that it is mainly generated through P-type Ca2+ channel s.