Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.
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 mM) and 10-100 microM [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 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.
Ismailov
I I
II
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Berdiev
B K
BK
Benos
D J
DJ
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000187:drug effects; D017136:Ion Transport / Q000502:physiology; D007668:Kidney / Q000187:drug effects*; D008051:Lipid Bilayers; D012964:Sodium / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects*; D013997:Time Factors
D008051:Lipid Bilayers; D015222:Sodium Channels; D012964:Sodium; D002118:Calcium