Loop Gating of Connexin Hemichannels Involves Movement of Pore-lining Residues in the First Extracellular Loop Domain

Unapposed connexin hemichannels exhibit robust closure in response to membrane hyperpolarization and extracellular calcium. This form of gating, termed “loop gating,” is largely responsible for regulating hemichannel opening, thereby preventing cell damage through excessive flux of ions and metabolites. The molecular components and structural rearrangements underlying loop gating remain unknown. Here, using cysteine mutagenesis in Cx50, we demonstrate that residues at the TM1/E1 border undergo movement during loop gating. Replacement of Phe⁴³ in Cx50 with a cysteine resulted in small or no appreciable membrane currents. Bath application of dithiothreitol or TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine), reagents that exhibit strong transition metal chelating activity, led to robust currents indicating that the F43C substitution impaired hemichannel function, producing “lock-up” in a closed or poorly functional state due to formation of metal bridges. In support, Cd²⁺ at submicromolar concentrations (50–100 nm) enhanced lock-up of F43C hemichannels. Moreover, lock-up occurred under conditions that favored closure, indicating that the sulfhydryl groups come close enough to each other or to other residues to coordinate metal ions with high affinity. In addition to F43C, metal binding was also found for G46C, and to a lesser extent, D51C substitutions, positions found to be pore-lining in the open state using the substituted-cysteine accessibility method, but not for A40C and A41C substitutions, which were not found to reside in the open pore. These results indicate that metal ions access the cysteine side chains through the open pore and that closure of the loop gate involves movement of the TM1/E1 region that results in local narrowing of the large aqueous connexin pore.Connexins are a large family of homologous integral membrane proteins that form gap junction (intercellular) channels that provide a direct communication pathway between neighboring cells. Gap junctions are formed by the docking of two hemichannels, which themselves can function in an undocked or unapposed configuration as ion channels that signal across the plasma membrane. Each hemichannel is composed of a hexamer of connexin subunits. The accepted membrane topology of a connexin subunit has four transmembrane domains (TM1–TM4)³ and two extracellular loops (E1 and E2) with amino and carboxyl termini located intracellularly (reviewed in Ref. 1).Connexin cell-cell channels and hemichannels are voltage dependent and two distinct voltage-sensitive gating mechanisms appear to be built into each hemichannel (2). One gating mechanism proposed to be located at the cytoplasmic end of the hemichannel is termed V_j gating, a name derived from studies of gap junction (cell-cell) channels describing sensitivity to transjunctional voltage, V_j, the voltage difference between coupled cells. The other gating mechanism is putatively ascribed to the extracellular end of the hemichannel and has been provisionally termed loop gating, because of the resemblance of gating transitions to those associated with initial opening of newly formed cell-cell channels (3, 4), a process that conceivably involves the extracellular loop domains.Loop gating is a robust gating mechanism that together with extracellular divalent cations, principally Ca²⁺, is largely responsible for keeping unapposed hemichannels closed at resting membrane potentials (5). Reports have suggested that extracellular divalent cations act as gating particles that enter and block the pore upon hyperpolarization (6, 7). An alternative model was recently proposed whereby extracellular divalent cations act as modulators of loop gating, an intrinsically voltage-sensitive mechanism, by stabilizing the closed conformation and shifting activation such that opening occurs at more positive potentials (8).Although loop gating plausibly involves conformational changes associated with the extracellular loops, molecular components underlying loop gating as well as the location of the putative gate remain unknown. A recent study using chick homologues to the mammalian connexins, Cx46 and Cx50, reported that two charged residues were important determinants of the different gating characteristics exhibited by these two connexin hemichannels (9). The implicated residues are at position 9 located in the NH₂-terminal domain and position 43 in the E1 domain. In Cx46 hemichannels, Glu⁴³ and other flanking residues at the TM1/E1 border (Ala³⁹, Gly⁴⁶, and Asp⁵¹) were shown to reside in the aqueous pore in the open state (10). Because it is likely that domains involved in permeation and gating of connexin channels are closely linked (reviewed in Ref. 11), we examined whether these residues are involved in structural rearrangements associated with loop gating. In this study, we engineered cysteines at residues in the TM1/E1 border in Cx50 hemichannels and used the ability of sulfhydryl groups to form disulfide bonds and/or to complex with heavy metal ions to report conformational changes that occur during gating.