Characterization of the Ca²⁺ current in freshly dissociated crustacean peptidergic neuronal somata

Janet E. Richmond, Emanuele Sher and Ian M. Cooke
J. Neurophysiol. 73:2357-2368(1995)

Summary and Conclusions

1. Freshly dissociated neuronal somata of the crab (Cardisoma carnifex) X-organ were studied in the whole-cell patch clamp configuration. In order to characterize the Ca²⁺ currents in these somata, recordings were made under conditions designed to suppress K⁺ and Na⁺ currents.
2. In 52 mM external Ca²⁺ the threshold for activation of Ca²⁺ currents was above -40 mV, with peak amplitudes occurring around +10 to +20 mV. The full component of the current was available for activation at -50 mV since no current increase was observed when the holding potential was increased to -90 mV. These characteristics of the current characterize it as a high-voltage activated (HVA) current.
3. The Ca²⁺ current was almost completely (60-90%) inactivated within 200 ms at maximal current potentials (+10 to +20 mV). The decay was best described by a double-exponential function with a fast and slow component of inactivation (τ_f = 12 ms and τ_s = 64 ms). Both Sr²⁺ and Ba²⁺ substitutions reduced the rates of inactivation.
4. In double-pulse experiments, plots of variable prepulse potential vs. test pulse current produced a U-shaped curve with test pulse currents showing maximal inactivation at potentials which produced maximal Ca²⁺ influx during the prepulse. Tail currents also displayed a U-shaped inactivation curve. The extent of current-dependent inactivation was sequentially reduced by Sr²⁺ and Ba²⁺ substitutions. These data suggest that inactivation in crab somata is predominantly Ca²⁺-dependent. The remaining inactivation of Ba²⁺ currents suggests that there is also a component of voltage-dependent inactivation in the somata.
5. Part of the inactivated Ca²⁺ current could be recovered during short (4-10 ms) hyperpolarizing pulses to -130 mV. The absolute extent of recovery from inactivation was greatest for currents carried by Ca²⁺ rather than Sr²⁺ or Ba²⁺. When voltage-dependent inactivation was dominant (Ba²⁺ currents), the relative amount of current recovered was greater. The data suggest that hyperpolarizing pulses are more effective in removing voltage-dependent inactivation, but also allow some recovery from Ca²⁺-dependent inactivation.
6. In the crab saline, which contained 24 mM Mg²⁺, the amplitudes of currents carried by 52 mM Ca²⁺, Sr²⁺ and Ba²⁺ were similar. Removing the Mg²⁺ from the saline augmented both the Ba²⁺ and Sr²⁺ currents relative to the Ca²⁺ current. The dose-response relationship between Mg²⁺ concentration and current amplitude was compared for 52 mM Ca²⁺, Sr²⁺ and Ba²⁺. Mg²⁺ blocked Ba²⁺>Sr²⁺>Ca²⁺. The ability of Mg²⁺ to suppress HVA currents was also dependent on the concentration of permeant divalent ions used.
7. The ability of several known inorganic Ca²⁺ channel blockers to effect Ca²⁺ current amplitude was determined. The order of blocking potency was La³⁺=Cd²⁺>Ni²⁺=Co²⁺>Mg²⁺.
8. No effects on Ca²⁺ current amplitude were found with nifedepine (µ10 M), Bay K 8644 (1 µM), ω-conotoxin GVIA, ω-Agatoxin IVA or ω-conotoxin MVIIC, indicating that the HVA Ca²⁺ current in X-organ somata is pharmacologically distinct from other characterized channels of the L-, N-, P-, and Q-types.

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