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Article

Canhui Li^*, Sylvie Breton, Rebecca Morrison, Carolyn L. Cannon^||, Francesco Emma^||, Roberto Sanchez-Olea^||, Christine Bear^*, and Kevin Strange

From the ^* Division of Cell Biology, Research Institute, Hospital for Sick Children and Physiology Department, University of Toronto, Toronto, Ontario, Canada M5G 1X8; Renal Unit, Massachusetts General Hospital, Boston, Massachusetts 02129; Anesthesiology Research Division, Department of Anesthesiology and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and ^|| Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

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.

Key Words: cell volume • swelling-activated anion channels • planar lipid bilayer • liposomes • recombinant protein

Abbreviations: CKII, casein kinase II; GST, glutathione S-transferase; NMDG, N-methyl-D-glucamine

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