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.
Article|
September 01 1995
Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.
I I Ismailov,
I I Ismailov
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
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B K Berdiev,
B K Berdiev
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
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D J Benos
D J Benos
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
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I I Ismailov
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
B K Berdiev
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
D J Benos
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Online ISSN: 1540-7748
Print ISSN: 0022-1295
J Gen Physiol (1995) 106 (3): 445–466.
Citation
I I Ismailov, B K Berdiev, D J Benos; Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.. J Gen Physiol 1 September 1995; 106 (3): 445–466. doi: https://doi.org/10.1085/jgp.106.3.445
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