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 cu ltures prepared from the small intestines of neonatal or young adult r ats were studied with tight seal whole-cell recordings. The kinetic an d pharmacological properties of these neurons were analyzed. 2. All ne urons had rapidly inactivating, tetrodotoxin (TTX)-sensitive Na+ curre nts that could be evoked by steps to potentials more positive than -50 mV. Holding potentials more negative than -65 mV were necessary to re move steady-state inactivation. No TTX-insensitive Na+ currents were o bserved, thus the ability of subsets of myenteric neurons to fire acti on 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 TT X and antagonists of K+ channels. Currents were significantly larger w hen Ba2+ replaced Ca2+ as the charge carrier. Cd2+ and Gd3+ blocked Ca 2+ and Ba2+ currents rapidly and reversibly. High-voltage-activated (H VA) Ca2+ and Ba2+ currents were observed in all neurons. Too few neuro ns possessed detectable low-voltage-activated Ca2+ currents to permit detailed study. 4. HVA Ca2+ and Ba2+ currents evoked from holding pote ntials 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 o f 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 indis tinguishable rates of deactivation: a single time constant was suffici ent to fit the decay of tail currents. The relative amplitudes of the two components varied considerably among different neurons. 5. Ca2+ an d Ba2+ currents could be divided into two pharmacologically distinct c omponents on the basis of sensitivity to omega-conotoxin GVIA (I(omega CgTX)) and to dihydropyridine antagonists (I(DHP)). At holding potenti als more positive than -70 mV, a combination of omegaCgTX and DHPs com pletely blocked Ca2+ and Ba2+ currents in most neurons. At holding pot entials more negative than -50 mV, I(omegaCgTX)) and I(DHP) each conta ined decaying and sustained components. I(omegaCgTx) activated more sl owly than did I(DHP). The DHP agonist Bay K 8644 increased the amplitu de of I(DHP) and slowed its rate of deactivation. 6. The results sugge st that myenteric neurons may have as few as two subtypes of HVA Ca2channels: 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 n ormal resting membrane potentials of these neurons lie within the stee p regions of the inactivation curves of their Na+ and Ca2+ currents, r elatively small changes in resting membrane potential, such as those c aused by slow synaptic potentials, should significantly affect the amp litude of inward currents flowing during action potentials.