Recombinant pI(Cln) forms highly cation-selective channels when reconstituted into artificial and biological membranes

L. I. Canhui, Sylvie Breton, Rebecca Morrison, Carolyn L. Cannon, Francesco Emma, Roberto Sanchez-Olea, Christine Bear, Kevin Strange

Research output: Contribution to journalArticlepeer-review

Abstract

pI(Cln) has been proposed to be the swelling-activated anion channel responsible for I(Cl,swell), or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pI(Cln) into artificial and biological membranes. Single channels were observed when pI(Cln) was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by -31.2 ± 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl- with glutamate, but was undetectable when K+ was replaced by N-methyl-d- glucamine. Reconstitution of pI(Cln) into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pI(Cln) with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pI(Cln) inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pI(Cln) and not minor contaminant proteins. However, these findings do not support the hypothesis that pI(Cln) is the anion-selective I(Cl,swell) channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pI(Cln), or may be a consequence of in vitro reconstitution of purified, recombinant protein.

Original languageEnglish
Pages (from-to)727-736
Number of pages10
JournalJournal of General Physiology
Volume112
Issue number6
DOIs
Publication statusPublished - 1998

Keywords

  • Cell volume
  • Liposomes
  • Planar lipid bilayer
  • Recombinant protein
  • Swelling-activated anion channels

ASJC Scopus subject areas

  • Physiology

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