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Department of Cell Biology and the Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322

Address correspondence to Criss Hartzell, Department of Cell Biology, Emory University School of Medicine, 615 Michael St., Whitehead Building 535, Atlanta, GA 30322-3030. Fax: (404) 727-6256; email: criss.hartzell{at}emory.edu

Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed various mutant constructs of mBest2 in HEK-293 cells to explore the contributions to anion selectivity of serine-79 and other amino acids (V78, F80, G83, F84, V86, and T87) located in the putative second transmembrane domain (TMD2). Residues selected for mutagenesis were distributed throughout TMD2, but mutations at all positions changed the selectivity. The effects on selectivity were rather modest. Replacement of residues 78, 79, 80, 83, 84, 86, or 87 with cysteine had similar effects: the permeability of the channel to SCN relative to Cl (P_SCN/P_Cl) was decreased three- to fourfold and the relative SCN conductance (G_SCN/G_Cl) was increased five- to tenfold. Side chains at positions 78 and 80 appeared to be situated close to the permeant anion, because the electrostatic charge at these positions affected permeation in specific ways. The effects of charged sulfhydryl-reactive MTS reagents were the opposite in the V78C and F80C mutants and the effects were partially mimicked by substitution of F80 with charged amino acids. In S79T, switching from Cl to SCN caused slow changes in G_SCN/G_Cl ( = 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked.

Key Words: chloride channels • ion permeation • electrophysiology • mutagenesis • ion channel

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