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

pI(Cln) has been proposed to be the swelling-activated anion channel respon sible for I-Cl,I- swell, or a channel regulator. We tested the anion channe l hypothesis by reconstituting recombinant pI(Cln) into artificial and biol ogical membranes. Single channels were observed when pI(Cln) was reconstitu ted into planar lipid bilayers. In the presence of symmetrical 300 mM KCI, 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 C l- with glutamate, but was undetectable when K+ was replaced by N-methyl-d- glucamine. Reconstitution of pI(Cln) into liposomes increased Rb-86(+) upta ke by three- to fourfold, but had no effect on Cl-36(-) uptake. Phosphoryla tion of pI(Cln) with casein kinase II or mutation of Gig, G56, and G58 to a lanine decreased channel open probability and Rb-86(+) uptake. When added t o the external medium bathing Sf9 cells, pI(Cln) inserted into the plasma m embrane and increased cell cation permeability. Taken together, these obser vations demonstrate that channel activity is due to pI(Cln) and not minor c ontaminant proteins. However, these findings do not support the hypothesis that pI(Cln) is the anion-selective I-Cl,I- swell channel. The observed cat ion channel activity may reflect an as yet to be defined physiological func tion of pI(Cln), or may be a consequence of in vitro reconstitution of puri fied, recombinant protein.