Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Abstract

The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances of Necturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (Vmc) results in a marked decrease in apical membrane fractional resistance (fRa), whereas hyperpolarization of Vmc results in either no change in fRa or a small increase, and b) the voltage-dependent changes in fRa are essentially complete within 500 msec. Exposure of the tissue to 5 mM TEA+ on the mucosal side caused no significant change in baseline Vmc (-69 +/- 2 mV) and yet virtually abolished the voltage dependence of fRa. A possible interpretation of these results is that two types of K+ channels exist in the apical membrane, with different voltage dependencies and TEA+ sensitivities. Acidification or Ba2+ addition to the mucosal solution also reduced the voltage-dependent changes in fRa. The time courses of the changes in fRa and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (+/- 200 microA/cm2). No secondary change in fRa was observed with mucosa-to-serosa currents, but a slow TEA+-sensitive decrease in fRa (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (less than 500 msec) voltage-dependent decrease in fRa is due to a fall in apical membrane resistance. The later decrease in fRa is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimating fRa than the current-induced effects on the lateral intercellular spaces. In principle, TEA+ can be used to prevent the nonlinear behavior of Ra during measurements of the voltage divider or membrane resistance ratio.

Effects of changes in mucosal solution Cl- or K+ concentration on cell water volume of Necturus gallbladder epithelium.

“…The resulting voltage deflections (measured at 800 ms from the onset of the pulse) were corrected for series solution resistances and used to calculate transepithelial resistance (R,) and the apparent ratio of cell membrane resistances (R JR b = AVmc /AV~) (see Stoddard and Reuss, 1988b). Transepithelial voltage (Vm~), cell membrane voltages (V,,~ and V,), and differential ion-sensitive electrode voltages (Vio .…”

“…Intracellular microelectrode (Stoddard and Reuss, 1988b) and patch-clamp studies (Segal and Reuss, 1990a, b) suggest the existence of at least two types of apical K ÷ conductances, one of which is a maxi K + channel gated by membrane depolarization and by elevation of [Ca2÷]i . This pathway can be almost totally blocked by TEA ÷.…”

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.

“…[n a separate set of studies, G. was estimated as a function of Vm, using 1-s transepithelial current clamps in the range _+ 300 #A/cm 2 (Stoddard and Reuss, 1988b (Stoddard and Reuss, 1988a). Recent work has revealed that depolarizing Vm, decreases Ra (Garcia-Diaz et ai., 1983;Stoddard and Reuss, 1988b).…”

“…Recent work has revealed that depolarizing Vm, decreases Ra (Garcia-Diaz et ai., 1983;Stoddard and Reuss, 1988b). In the Appendix, we refine the procedure for estimating RJRb from AV,,~/AV~, measurements, accounting for voltage-induced changes in Ga and E a.…”

“…Our major goal is to assess the contribution of these channels to the resting apical membrane K + conductance (G~), and to the increase in Ga elicited by apical membrane depolarization (Garcia-Diaz et al, 1983;Stoddard and Reuss, 1988b). We develop simple models to describe the steady-state gating and conduction properties of single maxi K § channels, and use these models as a basis to draw quantitative comparisons with the apical membrane conductance, assessed using intracellular microelectrode techniques.…”