Activation of calcium-dependent chloride channels in rat parotid acinar cells
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abstract
The Ca2%2b and voltage dependence Ca2%2b-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp technique. Acinar cells were dialyzed with buffered free Ca2%2b concentrations ([Ca2%2b](i)) from <1 nM to 5 μM. Increasing [Ca2%2b](i) induced na increase in Cl- current at all membrane potentials. In cells dialyzed with [Ca2%2b](i) >25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential component. The steady-state current-voltage relationship showed outward rectification at low [Ca2%2b](i) but became more linear as the [Ca2%2b](i) increased because of a shift in Cl- channel activation toward more negative voltages. The Ca2%2b dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparent K(d) and Hill coefficient obtained from this analysis were both functions of membrane potential. The K(d) decreased from 417 to 63 nM between -106 and %2b94 mV, whereas the Hill coefficient was always >1 and increased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channel steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2%2b binding steps before a final Ca2%2b-independent transition to the conducting conformation. Channel activation proceeds sequentially through three closed states before reaching the open state. The Ca2%2b binding steps of this model have a voltage dependence similar to that of the K(d) from the Hill analysis. The simplest interpretation of our findings is that these channels are directly activated by Ca2%2b ions that bind to sites ~13%25 into the membrane electric field from the cytoplasmic surface.
