Structural and functional characterization of a hyperpolarization-activated Cl- channel, CLC-2

초록

The CLC family proteins are crucial membrane proteins responsible for maintaining chloride ions homeostasis across the biological membranes. Among them, four CLCs (CLC-1, -2, -K1, and -K2) are Cl- channels, but others (CLC-3 to -7) are Cl-/H+ antiporters. Interestingly, previous studies have shown that CLC channels respond differently to the transmembrane voltages: CLC-1 is activated by membrane depolarization, CLC-2 is a hyperpolarization activated channel. A conserved glutamate residue has been suggested to confer the voltage-dependency of both CLC-1 and CLC-2 channels. However, the mechanism by which this highly conserved glutamate residue senses transmembrane voltage in opposite directions remains largely unknown. To gain an insight into the voltage-dependent mechanism of CLC channels, we examined the voltage-dependent activation of mouse CLC-2 channels by using whole-cell patch-clamp recordings. And to further elucidate the structural mechanism of voltage sensing, we determined the cryo-EM structure of wild-type mCLC-2 at approximately 3 Å resolution. We are currently optimizing cryo-EM conditions to obtain a higher resolution structure as well as structure of mutant channels.