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Contribution of Cytosolic Cysteine Residues to the Gating Properties of the Kir2.1 Inward Rectifier

Département de physiologie, Groupe de recherche en transport membranaire, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7

Correspondence: Address reprint requests to Dr. Rémy Sauvé, Groupe de recherche en transport membranaire, Département de physiologie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec, Canada H3C 3J7. E-mail: remy.sauve{at}umontreal.ca.

The topological model proposed for the Kir2.1 inward rectifier predicts that seven of the channel 13 cysteine residues are distributed along the N- and C-terminus regions, with some of the residues comprised within highly conserved domains involved in channel gating. To determine if cytosolic cysteine residues contribute to the gating properties of Kir2.1, each of the N- and C-terminus cysteines was mutated into either a polar (S, D, N), an aliphatic (A,V, L), or an aromatic (W) residue. Our patch-clamp measurements show that with the exception of C76 and C311, the mutation of individual cytosolic cysteine to serine (S) did not significantly affect the single-channel conductance nor the channel open probability. However, mutating C76 to a charged or polar residue resulted either in an absence of channel activity or a decrease in open probability. In turn, the mutations C311S (polar), C311R (charged), and to a lesser degree C311A (aliphatic) led to an increase of the channel mean closed time due to the appearance of long closed time intervals (T_c 500 ms) and to a reduction of the reactivation by ATP of rundown Kir2.1 channels. These changes could be correlated with a weakening of the interaction between Kir2.1 and PIP₂, with C311R and C311S being more potent at modulating the Kir2.1-PIP₂ interaction than C311A. The present work supports, therefore, molecular models whereby the gating properties of Kir2.1 depend on the presence of nonpolar or neutral residues at positions 76 and 311, with C311 modulating the interaction between Kir2.1 and PIP₂.

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