Characterization of Na+ and Ca2+ currents in bag cells of sexually immature Aplysia californica

Abstract

The neurosecretory bag cells of sexually mature Aplysia californica release egg-laying hormones as part of the reproductive process after a train of action potentials termed afterdischarge. Whole-cell voltage-clamp experiments were performed in cultured cells from sexually immature A. californica to characterize the inward voltage-gated currents for Na+ and Ca2+. The goal of these experiments was to investigate the regulation of excitability during sexual maturation. Na+ currents in bag cells of immature A. californica were similar in several ways to those of mature animals. The Na+ currents activated at voltages less negative than -30 mV and peaked at 10-20 mV in artificial sea water. The time course and pharmacology of bag cell Na+ currents were similar to those of bag cells from mature A. californica, although the Na+ current density was lower in immature A. californica. Na+ currents were inhibited by tetrodotoxin (50 nmol l-1). The Na+ current was relatively insensitive to depolarized holding potentials (V(h)), maintaining approximately 50% of peak current amplitude present at V(h)=-70 mV throughout the activation range at V(h)=-30 mV. In experiments using a 1 s depolarized V(h) prior to a test pulse, the half-inactivation voltage (v( 1/4 )) was -27 mV. Recovery of immature Na+ current from steady-state inactivation at V(h)=-30 mV had a time constant (τ) of 9.5 ms, significantly slower than in mature animals. Ca2+ currents of immature A. californica activated at approximately -30 mV and peaked at approximately 20 mV with 11 mmoll-1 Ba2+ as the charge carrier. The principle differences from mature Ca2+ currents were the low density of the immature Ca2+ currents and their 'run-down' in whole-cell recordings. The pharmacology and V( 1/4 ) of bag cell Ca2+ currents were similar to those of L-type Ca2+ currents in mature cells. The Ca2+ currents were inhibited 61 ± 10% by nifedipine (10 μmoll-1) and were unaffected by ω-conotoxin GVIA (10 μmoll-1). The Ca2+ currents were relatively insensitive to depolarized V(h), activating maximally at V(h)=-90, -70 and -50 mV, and maintaining 50% of this peak current amplitude throughout the activation range at V(h)=-30 mV. The V( 1/4 ) was -23 mV in experiments in which cells were subjected to a 1 s depolarized V(h) prior to a test pulse. Na+ current amplitudes were maintained or increased during 1 min of 4 Hz test pulses in bag cells at V(h)=-70 mV and V(h)=-30 mV. In contrast, Ca2+ current run-down occurred during 1 min of 4 Hz test pulses in seven of 10 cells at V(h)=-70 mV and in 12 of 12 cells at V(h)=-30 mV. The observed scarcity of Na+ and Ca2+ currents in immature bag cells as well as the specific characteristics of immature bag cell Ca2+ currents make repetitive action potential firing and hormone release less likely than in mature bag cells.