Connexin hemichannels, the first identified eukaryotic large-pore channels, mediate the uncoupled permeation of both atomic ions and small molecules. Small-molecule permeation exhibits voltage dependence, saturability, and selectivity, revealing transporter-like features of these channels. To investigate the molecular basis of voltage-dependent transport, we combined two-electrode voltage clamp (TEVC) recordings, which allow precise control of membrane potential and measurement of ion currents, with DAPI uptake assays to monitor small-molecule transport in real-time in Cx26 hemichannels. Consistent with the uncoupling of ion and small-molecule permeation, DAPI transport followed a Boltzmann function, showing increased uptake at negative potentials and complete suppression at positive potentials, which is opposite to the trend observed for ion conduction. Molecular dynamics simulations identified an extracellular binding site that transiently traps and releases DAPI in a voltage-dependent manner. Specifically, an acidic vestibular pocket at the extracellular entrance, formed by Asp46, Glu47, and Asp50, engages the +2 charge of DAPI. Positive voltage strengthened binding within this pocket, stabilizing the bound state and suppressing translocation. Vestibular occupancy depended on both the duration of depolarization and the concentration of DAPI. Mutations of Glu47 or Asp50 but not Asp46, reduced vestibular accumulation and flattened the voltage dependence of transport, consistent with a voltage-tuned trap-release step that limits transport rate. Together, these findings support a hybrid channel/transporter model for Cx26, in which small-molecule transport is governed by voltage-dependent binding and unbinding at a discrete extracellular site, mechanistically separable from ion conduction.
Gaete et al. (Sun,) studied this question.
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