Connexin hemichannels and cell-cell channels: comparison of properties

Abstract

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 (~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 Ca 2+ 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.

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

“…An AFM equipped with a buffer chamber allowed us to observe Cx26 membranes under native conditions at high resolution. Analogous to the TSF system of Harris et al (37) or the excised patch experiments of Verselis et al (38), this is a reductionist system. We limit the number of variables in the imaging experiment, which makes our results complementary to the pH studies done in whole cells (12,39).…”

“…For cardiac connexins (Cx43, Cx40, and Cx45), Delmar and co-workers (12) demonstrated that truncation of the C terminus significantly decreased the response of the intact channels to acidification. In a study comparing Cx46 channels and hemichannels expressed in oocytes, and using excised patches of Cx46 hemichannels, Verselis and coworkers (19,38) showed that Cx46 hemichannels act similarly to intact channels and close reversibly with acidification. The site of action for H ϩ is localized to the cytoplasmic domains.…”

New roles for astrocytes: Gap junction hemichannels have something to communicate

“…An open hemichannel could well exhibit asymmetry in fixed charges that causes rectification, whereas the asymmetry is neutralized in the cell-cell channel [45,51].…”

Sharing signals: connecting lung epithelial cells with gap junction channels

“…Given this, connexin hemichannels may release ATP as part of an apoptosis pathway. Consistent with this possibility, Cx46 hemichannels open under physiological conditions (213,268,282), an intriguing notion given the upregulation of Cx46 in response to lung injury (described below). The notion of functional roles for connexin hemichannels is being further explored, as this is still a controversial area in need of further study.…”

“…This has led to the suggestion that connexin hemichannels at the cell surface might mediate ATP release directly from the cytosol to the extracellular environment (54,257). Permeability from connexin hemichannels, by and large, seems to reflect the permeability of complete gap junction channels and thus could certainly enable ATP diffusion (88,171,268,282). However, it is still feasible that cells might use a different plasma membrane channel for mediating ATP release that nonetheless requires cell-cell coupling through gap junctions.…”