VOLTAGE-DEPENDENT SODIUM AND CALCIUM CURRENTS OF RAT MYENTERIC NEURONS IN CELL-CULTURE

1. Inward currents of myenteric neurons that had been grown in cell cultures prepared from the small intestines of neonatal or young adult rats were studied with tight seal whole-cell recordings. The kinetic and pharmacological properties of these neurons were analyzed. 2. All neurons had rapidly inactivating, tetrodotoxin (TTX)-sensitive Na+ currents that could be evoked by steps to potentials more positive than -50 mV. Holding potentials more negative than -65 mV were necessary to remove steady-state inactivation. No TTX-insensitive Na+ currents were observed, thus the ability of subsets of myenteric neurons to fire action potentials in TTX must depend upon their density of Ca2+ channels. 3. Ca2+ and Ba2+ currents were studied in neurons perfused internally with CsCl to block K+ currents and bathed with solutions containing TTX and antagonists of K+ channels. Currents were significantly larger when Ba2+ replaced Ca2+ as the charge carrier. Cd2+ and Gd3+ blocked Ca2+ and Ba2+ currents rapidly and reversibly. High-voltage-activated (HVA) Ca2+ and Ba2+ currents were observed in all neurons. Too few neurons possessed detectable low-voltage-activated Ca2+ currents to permit detailed study. 4. HVA Ca2+ and Ba2+ currents evoked from holding potentials more negative than -50 mV could be divided into two kinetically distinguishable components with very different rates of inactivation. A ''decaying'' component inactivated relatively rapidly with a t1/2 of 25-75 ms. A ''sustained'' component inactivated quite slowly with a t1/2 of 1-5 s. At more positive holding potentials, only the sustained component was observed. Although the two kinetically distinguishable components had different current-voltage relationships, they had indistinguishable rates of deactivation: a single time constant was sufficient to fit the decay of tail currents. The relative amplitudes of the two components varied considerably among different neurons. 5. Ca2+ and Ba2+ currents could be divided into two pharmacologically distinct components on the basis of sensitivity to omega-conotoxin GVIA (I(omegaCgTX)) and to dihydropyridine antagonists (I(DHP)). At holding potentials more positive than -70 mV, a combination of omegaCgTX and DHPs completely blocked Ca2+ and Ba2+ currents in most neurons. At holding potentials more negative than -50 mV, I(omegaCgTX)) and I(DHP) each contained decaying and sustained components. I(omegaCgTx) activated more slowly than did I(DHP). The DHP agonist Bay K 8644 increased the amplitude of I(DHP) and slowed its rate of deactivation. 6. The results suggest that myenteric neurons may have as few as two subtypes of HVA Ca2+ channels: omegaCgTX-sensitive ones and DHP-sensitive ones. This simple classification requires that each of the channel subtypes be capable of existing in rapidly and slowly inactivating modes. 7. Because the normal resting membrane potentials of these neurons lie within the steep regions of the inactivation curves of their Na+ and Ca2+ currents, relatively small changes in resting membrane potential, such as those caused by slow synaptic potentials, should significantly affect the amplitude of inward currents flowing during action potentials.