1. N-type (ω-conotoxin sensitive) calcium currents (I(Ca)) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 ± 0.3 MΩ; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited I(Ca), which peaked ~20 mV and declined with increasing depolarizations. 2. Evidence for a low- threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (<100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage- activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half- activation voltage and slope factor of 2.9 and 7.7 mV for I(Ca), -13.1 mV and 7.8 for Ba2+ current (I(Ba)) and -2.3 mV and 7.8 for Sr2+ current (I(Sr)). The time course of activation was monotonic with time constant (τ) for I(Ca) ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (τ(fast) of 25 ± 4 ms; and τ(slow) of 473 ± 64 ms; mean ± SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of ~350 ms, I(Sr) showed inactivation whereas Ba2+ virtually removed the decay. However, I(Ba) turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for I(Ca), I(Ba), and I(Sr). Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(β-aminoethyl ether)- N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed. Series of experiments were conducted where relatively high Ca2+ (10-20 μM) was present in the pipette to saturate the Ca2+-induced process. Under this condition, a voltage-dependent inactivation became prominent. Inactivation of I(Ca) was dependent on both current and voltage.
|Original language||English (US)|
|Number of pages||13|
|Journal||Journal of Neurophysiology|
|State||Published - 1994|
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