Connexin hemichannels and cell-cell channels: comparison of properties

Connexin46 (Cx46) forms functional hemichannels in the absence of contact b y an apposed hemichannel and we have used these hemichannels to study gatin g and permeation at the single channel level with high time resolution. Usi ng both cell-attached and -excised patch configurations, we find that singl e Cx46 hemichannels exhibit some properties expected of half of a gap junct ion channel, as well as novel properties. Cx46 hemichannels have a large un itary conductance (similar to 300 pS) and a relatively large pore as inferr ed from permeability to TEA. Both monovalent cations and anions can permeat e, but cations are substantially more permeable. The open channel conductan ce shows marked inward rectification in symmetric salts. We find that the c onductance and permeability properties of Cx46 cell-cell channels can be ex plained by the series addition of two hemichannels. These data suggest that the pore structures of unapposed hemichannels and cell-cell channels are c onserved. Also like cell-cell channels, unapposed Cx46 hemichannels are clo sed by elevated levels of H+ or Ca2+ ions on the cytoplasmic face. Closure occurs in excised patches indicating that the actions of these agents do no t require a soluble cytoplasmic factor. Fast (<0.5 ms) application of H+ to either side of the open hemichannel causes an immediate small reduction in unitary conductance followed by complete closure with latencies that are d ependent on H+ concentration and side of application; sensitivity is much g reater to H+ on the cytoplasmic side. Closure by cytoplasmic H+ does not re quire that the hemichannel be open. Thus, H+ ions readily permeate Cx46 hem ichannels, but at high enough concentration close them by acting at a cytop lasmic site(s) that causes a conformational change resulting in complete cl osure. Extracellular H+ may permeate to act on the cytoplasmic site or act on a lower affinity extracellular site. Thus, the unapposed hemichannel is a valuable tool in addressing fundamental questions concerning the operatio n of gap junction channels that are difficult to answer by existing methods . The ability of Cx46, and perhaps other connexins, to form functional unap posed hemichannels that are opened by moderate depolarization may represent an unexplored role of connexins as mediators of transport across the plasm a membrane.