Recombinant pICln forms highly cation-selective channels when reconstituted into artificial and biological membranes.
pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln 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 pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pICln 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, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.
Li
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Division of Cell Biology, Research Institute, Hospital for Sick Children and Physiology Department, University of Toronto, Toronto, Ontario, Canada M5G 1X8.
Breton
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Morrison
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Cannon
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Emma
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Sanchez-Olea
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Bear
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Strange
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D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004285:Dogs; D066298:In Vitro Techniques; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008564:Membrane Potentials; D008567:Membranes, Artificial; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D018118:Chloride Channels; C498729:Clns1a protein, rat; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008567:Membranes, Artificial; D011994:Recombinant Proteins