Site-directed mutations in the transmembrane domain M3 of human connexin37alter channel conductance and gating

Connexin37 (Cx37) is expressed principally in endothelial cells. We have in troduced individual point mutations (Cx37-V156D or Cx37-K162E) in the putat ive pore lining segment M3 of a polymorphic human Cx37 (Cx37-S319) and expr essed them in N2A and RIN cells. RT-PCR and immunofluorescence microscopy w ere used to confirm the expression of the proteins. Stably transfected cell s were subjected to electrophysiological studies. Experiments were performe d on cell pairs using the dual whole cell patch-clamp method. Single channe l records showed that both mutants display a variety of conductive states ( Cx37-V156D, 47-250 pS; Cx37-K162E, 58-342 pS) in contrast to the typical hi gh conductance of 340-375 pS and subconductive state of 60-80 pS reported f or Cx37-S319. Analysis of the macroscopic data for Cx37-K162E revealed a br oadened Vo indicating the influence of the mutation on voltage gating. Our data indicate that substitution of a conserved residue with a charged resid ue could cause changes in the main state and/or in the size of the pore. It is possible that these particular residues in the M3 domain interact elect rostatistically with several of the other domains in the Cx37 protein. (C) 2001 Academic Press.