ACTIVATION OF CALCIUM-DEPENDENT CHLORIDE CHANNELS IN RAT PAROTID ACINAR-CELLS

The Ca2+ and voltage dependence of Ca2+-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp tech nique. Acinar cells were dialyzed with buffered free Ca2+ concentratio ns ([Ca2+](i)) from <1 nM to 5 mu M. Increasing [Ca2+](i) induced an i ncrease in Cl- current at all membrane potentials. In cells dialyzed w ith [Ca2+](i) >25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential comp onent. The steady-state current-voltage relationship showed outward re ctification at low [Ca2+](i) but became more linear as the [Ca2+](i) i ncreased because of a shift in Cl- channel activation toward more nega tive voltages. The Ca2+ dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparen t K-d and Hill coefficient obtained from this analysis were both funct ions of membrane potential. The K-d decreased from 417 to 63 nM betwee n -106 and +94 mV, whereas the Hill coefficient was always >1 and incr eased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channe l steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2+ binding steps be fore a final Ca2+-independent transition to the conducting conformatio n. Channel activation proceeds sequentially through three closed state s before reaching the open state. The Ca2+ binding steps of this model have a voltage dependence similar to that of the K-d from the Hill an alysis. The simplest interpretation of our findings is that these chan nels are directly activated by Ca2+ ions that bind to sites similar to 13% into the membrane electric field from the cytoplasmic surface.