Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (<30 raM) and 10-100 µM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans Buffering of the cis compartment [Ca2+] to nearly zero (<1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 ± 0.02 vs 0.02 ± 0.01 in control), but markedly reduced the influence of both cis- and trans- [Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCA2+] of 10-100 µM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 µM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na+ channels is indeed dependent upon the specific ionic environment surrounding the channels.
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