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. These proteins are expressed in various tissues and are conserved from bacteria to human. 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, and CLC-Ks are voltage-independent. A conserved glutamate residue (Gluex or Glugate) 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 (mCLC-2) by using whole-cell patch-clamp recordings. Based on the known structure of human CLC-1, we examined the mutational effects of putative pore-lining polar (T458 and Y561), negatively charged (E174 and E213), and positively charged residues (K212 and K568) on the voltage- and pH-dependent gating were examined. 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 structures of mutant channels that show altered voltage dependency.