Connexin domains relevant to the chemical gating of gap junction channels

Abstract

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both aminoand carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO 2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO 2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO 2 , whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL 2 ) of Cx38 was as sensitive to CO 2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO 2 sensitivity, but replacement of 5 arginines (R) with asparagines (N) at the beginning of CT (C 1 ) greatly enhanced the CO 2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C 1 maintain the CO 2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning

Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin

“…The possible role of CaM in the chemical gating of the β -family Cxs was first proposed by Peracchia and Wang [51]. CaM co-localizes with Cx32 [9] and directly gates Cx32-containing gap junctions [52].…”

Functional rescue of defective mutant connexons by pairing with wild-type connexons

“…Since essential regulatory domains for the opening and closing of gap junction channels are located at the COOH terminus of connexins [7,11,23,25], it was not surprising that these channels were defective in coupling. It was unexpected, however, that this defect in regulatory competence of a connexon is no longer detectable when a trCx45 connexon is paired with an intact connexon.…”

“…The permeability of these channels can be regulated by different mechanisms, such as changes in transjunctional voltage [1,29,31] or intracellular pH [11], but phosphorylation [20] and the binding of ligands [30] may also affect their conductance. The regulatory sites for these gating processes are presumably located at the COOH end [25] of the channel-forming protein, the connexin (Cx). A hexamer of these proteins forms one hemichannel, the connexon, which can pair with an identical connexon of a neighbouring cell, thus establishing a permeable homotypic cell-cell channel.…”

Structural basis for the selective permeability of channels made of communicating junction proteins

“…They initially postulated differential roles for the 1 st and 2 nd segments of the intracellular loop (CL 1 and CL 2 ) in V j -gating and CO 2 -induced (pH) gating, respectively. As deletion of most of the CT of Cx32 did not affect its CO 2 -sensitivity, but replacement of the arginines at the beginning of the CT (dubbed CT 1 ) with asparagines greatly increased CO 2 -gating, Peracchia and Wang [152] suggested that interaction occurs between negative residues in CL 1 with positive residues either in CT 1 or CL 2 , with the open channel resulting from CL 1 -CT 1 interaction and the chemically (pH, Ca) gated closed channel resulting from CL 1 -CL 2 interaction. Evidence that positive charges at CT 1 were key to the CO 2 -sensitivity, but not V j -sensitivity was presented [216] and incorporated into a model in which CT 1 acts as “a latch that keeps the channel open by immobilizing the CL, a potential gating element.” Breaking the CL 1 -CT 1 interaction would be part of the gating process that enables the CL (not CT) to move toward the channel pore and close the channel.…”