The N Terminus of Connexin37 Contains an α-Helix That Is Required for Channel Function

Abstract

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly ␣-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the ␣-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.

Hexadecameric structure of an invertebrate gap junction channel

“…An N-terminal deletion mutant of ShakBL, an innexin protein found in Drosophila, fails to induce conductance when paired homotypically, and induces much lower conductance when paired heterotypically with functional ShakBL [43]. These are consistent with the works on Cx37 showing that the N-terminal deletion mutants are mostly non-functional [44,45]. It is possible that the conformations of all purified INX-6 Δ N channels are fixed in a closed state by this truncation, which leads to the better crystallinity.…”

Specificity of the connexin W3/4 locus for functional gap junction formation

“…10 Bioinformatic secondary structure predictions and NMR studies suggest that the -group connexin NT domain possesses a predominantly -helical pore domain structure between positions W4 and S18. 30 indicate a 15% increase in  j and reduced cation selectivity associated with the Cx40 G2 mutation (Fig. 2), consistent with the interpretation of increased pore volume and less electrostatic charge in the vicinity of the amino terminus.…”

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure

“…However, all constructs containing at least 9 amino acids formed gap junction plaques, implying that the absolute length of the NT and the identities of specific amino acids are not requirements for normal biosynthesis of gap junctions. In agreement with these findings, Kyle et al [26] found that expression of a Cx37 mutant containing prolines at positions 10 and 15 (instead of the normal Leu15 and Gln15) also formed plaques. However, all of these mutations disrupted hemichannel and channel function.…”

“…It is interesting that the critical residues implicated in determining these various physiological properties are among the most divergent amino acids within the NT domain (see [26, 32, 35]). Although overall the sequence of this domain is quite conserved, the substitutions confer many of the characteristic properties that differentiate channels formed of different connexins.…”

“…The structure of the NT domain was initially investigated by studying synthetic peptides using circular dichroism and nuclear magnetic resonance (NMR) [26,53–55]. Based on the studies of a 15 amino acid peptide, Purnick et al [53] proposed a model for the NT of Cx26 with an α-helix extending from position 1 to 10 and a critical bend at positions 11 and 12 that was suggested to act as a “hinge” allowing the first 10 amino acids to swing into the pore and block the channel.…”