EVIDENCE FOR MULTIPLE OPEN STATES OF THE CA2-MUSCLE CELLS ISOLATED FROM THE GUINEA-PIG DETRUSOR( CHANNELS IN SMOOTH)

1. Whole-cell voltage clamp techniques were used to examine the proper ties of voltage-dependent Ca2+ channel currents in single smooth muscl e cells enzymatically dissociated from guinea-pig urinary bladder. Pot assium currents were blocked with intracellular Cs+. A holding potenti al of -60 mV was normally applied. 2. When the membrane potential was returned to the holding potential after a depolarizing step, tail curr ents were seen after depolarizations to positive potentials, and the s ize of the tail current increased with increasing positivity of the pr eceding depolarization. 3. After depolarization to +80 mV (a potential at which little inward current flowed through the Ca2+ channels) tail currents on returning to the holding potential increased in size as t he duration of the depolarization was increased.4. Investigation of th e mechanism mediating the tail currents showed that they were not flow ing through non-selective cation channels, and had no contribution fro m Ca2+-activated Cl- channels or Na+-Ca2+ exchange. 5. The tail curren ts and the inward currents evoked by a simple depolarizing test potent ial were equally decreased by nifedipine in a dose-dependent manner. T his suggests that L-type Ca2+ channels are responsible for both of the two types of inward currents. The inward currents were also inhibited in a similar manner when caffeine was applied. 6. Although the tail c urrents evoked on stepping from +80 mV to a holding potential of -60 m V increased in size with the duration of the conditioning potential, t he total membrane Ca2+ conductance did not increase, since the inward currents evoked on stepping to +20 mV (a potential at which the Ca2+ c hannels are still fully activated) did not change with time. 7. The am plitude of the inward current evoked by a simple depolarizing test pot ential was similar to that evoked on stepping to the same test potenti al after preconditioning at +80 mV, if the test potential was higher t han +20 mV. However, following repolarization to the holding potential , the amplitude of the subsequent tail current was larger and the deac tivation time constant longer, after the conditioning depolarization. These results suggest that the voltage-dependent Ca2+ channels have at least two open states with different time constants, the tail current being the result of a long open channel state induced by large depola rizations. 8. When variable repolarizing potentials were applied after n +80 mV depolarization (5 s), the current-voltage relationship of th e tail current was nearly linear between -60 and +30 mV. The deactivat ion was faster when a larger repolarization step was applied. 9. The r esults are consistent with the voltage-dependent Ca2+ channels in blad der smooth muscle cells having at least two available open channel sta tes, one rapidly deactivating and one slowly deactivating. The slowly deactivating state can be induced in a time-dependent manner by depola rization to positive potentials.