CHARACTERIZATION OF THE CA2+ CURRENT IN FRESHLY DISSOCIATED CRUSTACEAN PEPTIDERGIC NEURONAL SOMATA

1. Freshly dissociated neuronal somata of the crab (Cardisoma carnifex ) X-organ were studied in the whole cell patch-clamp configuration. 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 becau se no current increase was observed when the holding potential was inc reased to -90 mV. These characteristics of the current characterize it as a high-voltage activated (HVA) current. 3. The Ca2+ current was al most completely (60-90%) inactivated within 200 ms at maximal current potentials (+10 to +20 mV). The decay was best described by a double-e xponential function with a fast and slow component of inactivation ( t au(f) = 12 ms and tau(s) = 64 ms). Both Sr2+ and Ba2+ substitutions re duced the rates of inactivation. 4. In double-pulse experiments, plots of variable prepulse potential versus test pulse current produced a U -shaped curve with test pulse currents showing maximal inactivation at potentials that produced maximal Ca2+ influx during the prepulse. Tai l currents also displayed a U-shaped inactivation curve. The extent of current-dependent inactivation was sequentially reduced by Sr2+ and B a2+ substitutions. These data suggest that inactivation in crab somata is predominantly Ca2+ dependent. The remaining inactivation of Ba2+ c urrents suggests that there is also a component of voltage-dependent i nactivation in the somata. 5. part of the inactivated Ca2+ current cou ld 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-depen dent 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 t he crab saline, which contained 24 mM Mg2+, the amplitudes of currents carried by 52 mM Ca2+, Sr2+ and Ba2+ were similar. Removing the Mg2from the saline augmented both the Ba2+ and Sr2+ currents relative to the Ca2+ current. The dose-response relationship between Mg2+ concentr ation and current amplitude was compared for 52 mM Ca2+, Sr2+, and Ba2 +. Mg2+ blocked Ba2+ > Sr2+ > Ca2+. The ability of Mg2+ to suppress HV A currents was also dependent on the concentration of permeant divalen t ions used. 7. The ability of several known inorganic Ca2+ channel bl ockers to effect Ca2+ current amplitude was determined. The order of b locking potency was La3+ = Cd2+ > Ni2+ = Co2+ > Mg2+. 8. No effects on Ca2+ current amplitude were found with nifedepine(10 mu M), Bay K 864 4(1 mu M), omega-conotoxin GVIA, omega-agatoxin IVA, or omega-conotoxi n MVIIC, indicating that the HVA Ca2+ current in X-organ somata is pha rmacologically distinct from other characterized channels of the L, N, P, and Q types.