Protein kinase A phosphorylation and G protein regulation of purified renal Na+ channels in planar bilayer membranes.

I.I Ismailov,J.H Mcduffie,D.J Benos

doi:10.1016/s0021-9258(17)34052-8

I.I Ismailov, J.H Mcduffie + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)34052-8

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Abstract

Purified bovine renal epithelial Na+ channels incorporated into planar lipid bilayer membranes were used to evaluate the biophysical consequences of its phosphorylation by protein kinase A (PKA). We also studied the effects of pertussis toxin-induced ADP-ribosylation on single channel activity of nonphosphorylated and PKA-phosphorylated channels. PKA-induced phosphorylation resulted in a significant increase in single channel open probability (Po) with no change in single channel conductance, as well as increased the probability of multiple channel openings in the bilayer. Further, PKA conferred a voltage sensitivity to channel gating without affecting open channel conduction properties. PKA-phosphorylated Na+ channels were inhibited by subsequent ADP-ribosylation with pertussis toxin (PTX). Addition of guanosine 5'-3-O-(thio)triphosphate reversed this inhibition. However, exposure of nonphosphorylated Na+ channels to PTX increased channel open probability by a factor of 3-5. These results demonstrate that a cAMP-dependent pathway is an important regulatory element for amiloride-sensitive Na+ channels and that the effects of PTX-induced ADP-ribosylation of the channel-associated Gi protein on function depend upon the previous phosphorylation state of the protein.

Highlights

Purified bovine renal epithelial Na' channels incor- single channels incorporated into planar bilayers [13] and inporated into planar lipid bilayer membranes were used side-out and cell-attached membrane patches [14].a to evaluate the biophysical consequences of its phos- detailed biophysical analysis of the phosphorylated form of the phorylation by protein kinase A (PKA).We studied channel hasnot yet been undertaken
Directphosphorylation of purified and reconstituted renal Na' channel protein in planar lipid bilayers by PKA plus ATP increases theprobability of a single channel being in an open state butalso increases the frequency of multiple channel openings inthe membrane
Na' channel phosphorylation by PKA confers a voltage dependence to channel activity, such that the channopeel n probability is greaterat negative than positive appliedpotentials; Na' flows through the channel more readily at negative voltages

Summary

Introduction

Purified bovine renal epithelial Na' channels incor- single channels incorporated into planar bilayers [13] and inporated into planar lipid bilayer membranes were used side-out and cell-attached membrane patches [14].a to evaluate the biophysical consequences of its phos- detailed biophysical analysis of the phosphorylated form of the phorylation by protein kinase A (PKA).We studied channel hasnot yet been undertaken. The effects of pertussis toxin-induced ADP-ribosylation We have recently demonstrated thata GTP-binding protein, on single channel activity of nonphosphorylatedand Gui.S,associates with renal amiloride-sensitive Na'. Bubien et a1.' proposed a model recreversed this inhibition.,exposure of non- onciling thesedisparate observationsbased on experiments phosphorylatedNa' channels to PTX increased channel conducted in humanlymphocytes using thewhole cell configuopen probability by a factor of 3-5. These results dem- ration of the patch-clamptechnique. PTX onstrate that a cAMF"dependent pathway is an impor- would open nonphosphorylated Na' channels but inactivate tantregulatoryelement for amiloride-sensitive Na' previously phosphorylated ones, and CAMPtreatment would channels and thatthe effects of PTX-induced ADP-ribo- open Na' channels in theabsence of PTX treatment but close sylation of the channel-associatedGi protein on func- them in cells pretreated with P T X

Methods

Conclusion