RESUMO
Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemichannels or just hemichannels. Because some gap junction channels are permeable to negatively and/or positively charged molecules up to approximately 1kDa in size, it was thought that hemichannels should not open to the extracellular space. A growing amount of evidence indicates that opening of hemichannels does occur under both physiological and pathological conditions in astrocytes and other cell types. Electrophysiological studies indicate that hemichannels have a low open probability under physiological conditions but may have a much higher open probability under certain pathological conditions. Some of the physiological behaviours of astrocytes that have been attributed to gap junctions may, in fact, be mediated by hemichannels. Hemichannels constituted of Cx43, the main connexin expressed by astrocytes, are permeable to small physiologically significant molecules, such as ATP, NAD+ and glutamate, and may mediate paracrine as well as autocrine signalling. Hemichannels tend to be closed by negative membrane potentials, high concentrations of extracellular Ca2+ and intracellular H+ ions, gap junction blockers and protein phosphorylation. Hemichannels tend to be opened by positive membrane potentials and low extracellular Ca2+, and possibly by as yet unidentified cytoplasmic signalling molecules. Exacerbated hemichannel opening occurs in metabolically inhibited cells, including cortical astrocytes, which contributes to the loss of chemical gradients across the plasma membrane and speeds cell death.
Assuntos
Astrócitos/fisiologia , Junções Comunicantes/fisiologia , Conexinas/fisiologia , Eletrofisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Connexin46 (Cx46) forms functional hemichannels in the absence of contact by an apposed hemichannel and we have used these hemichannels to study gating and permeation at the single channel level with high time resolution. Using both cell-attached and -excised patch configurations, we find that single Cx46 hemichannels exhibit some properties expected of half of a gap junction channel, as well as novel properties. Cx46 hemichannels have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to TEA. Both monovalent cations and anions can permeate, but cations are substantially more permeable. The open channel conductance shows marked inward rectification in symmetric salts. We find that the conductance and permeability properties of Cx46 cell-cell channels can be explained by the series addition of two hemichannels. These data suggest that the pore structures of unapposed hemichannels and cell-cell channels are conserved. Also like cell-cell channels, unapposed Cx46 hemichannels are closed 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 not 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 dependent on H+ concentration and side of application; sensitivity is much greater to H+ on the cytoplasmic side. Closure by cytoplasmic H+ does not require that the hemichannel be open. Thus, H+ ions readily permeate Cx46 hemichannels, but at high enough concentration close them by acting at a cytoplasmic site(s) that causes a conformational change resulting in complete closure. 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 operation of gap junction channels that are difficult to answer by existing methods. The ability of Cx46, and perhaps other connexins, to form functional unapposed hemichannels that are opened by moderate depolarization may represent an unexplored role of connexins as mediators of transport across the plasma membrane.