Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.
The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.
Tang
Qingxiu
Q
Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Dowd
Terry L
TL
Verselis
Vytas K
VK
Bargiello
Thaddeus A
TA
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism; D017630:Connexins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000737:chemistry / Q000502:physiology; D015640:Ion Channel Gating; D008564:Membrane Potentials; D009865:Oocytes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism; D000097002:Gap Junction beta-1 Protein
D002104:Cadmium; D018031:Connexin 43; D017630:Connexins; D003545:Cysteine; D011993:Recombinant Fusion Proteins; D000097002:Gap Junction beta-1 Protein