Regulation of connexin hemichannel activity by membrane potential and the extracellular calcium in health and disease.

Connexins are thought to solely mediate cell-to-cell communication by forming gap junction channels composed of two membrane-spanning hemichannels positioned end-to-end. However, many if not all connexin isoforms also form functional hemichannels (i.e., the precursors of complete channels) that mediate the rapid exchange of ions, second messengers and metabolites between the cell interior and the interstitial space. Electrical and molecular signaling via connexin hemichannels is now widely recognized to be important in many physiological scenarios and pathological conditions. Indeed, mutations in connexins that alter hemichannel function have been implicated in several diseases. Here, we present a comprehensive overview of how hemichannel activity is tightly regulated by membrane potential and the external calcium concentration. In addition, we discuss the genetic mutations known to alter hemichannel function and their deleterious effects, of which a better understanding is necessary to develop novel therapeutic approaches for diseases caused by hemichannel dysfunction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Regulation of neuronal connexin-36 channels by pH.

Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the C- and N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH(i) since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions.

Molecular basis of voltage dependence of connexin channels: an integrative appraisal.

The importance of electrical and molecular signaling through connexin (Cx) channels is now widely recognized. The transfer of ions and other small molecules between adjacent cells is regulated by multiple stimuli, including voltage. Indeed, Cx channels typically exhibit complex voltage sensitivity. Most channels are sensitive to the voltage difference between the cell interiors (or transjunctional voltage, V(j)), while other channels are also sensitive to absolute inside-outside voltage (i.e., the membrane potential, V(m)). The first part of this review is focused on the description of the distinct forms of voltage sensitivity and the gating mechanisms that regulate hemichannel activity, both individually and as components of homotypic and heterotypic gap junctions. We then provide an up to date and precise picture of the molecular and structural aspects of how V(j) and V(m) are sensed, and how they, therefore, control channel opening and closing. Mutagenic strategies coupled with structural, biochemical and electrophysical studies are providing significant insights into how distinct forms of voltage dependence are brought about. The emerging picture indicates that Cx channels can undergo transitions between multiple conductance states driven by distinct voltage-gating mechanisms. Each hemichannel may contain a set of two V(j) gates, one fast and one slow, which mediate the transitions between the main open state to the residual state and to the fully closed state, respectively. Eventually, a V(m) gate regulates channel transitions between the open and closed states. Clusters of charged residues within separate domains of the Cx molecule have been identified as integral parts of the V(j) and V(m) sensors. The charges at the first positions of the amino terminal cytoplasmic domain determine the magnitude and polarity of the sensitivity to fast V(j)-gating, as well as contributing to the V(j)-rectifying properties of ion permeation. Additionally, important advances have been made in identifying the conformational rearrangements responsible for fast V(j)-gating transitions to the residual state in the Cx43 channel. These changes involve an intramolecular particle-receptor interaction between the carboxy terminal domain and the cytoplasmic loop.

Species specificity of mammalian connexin-26 to form open voltage-gated hemichannels.

Mutations of connexin-26 (Cx26) cause nonsyndromic hearing loss and other syndromes affecting ectoderm-derived tissues. While the exact mechanisms underlying these diseases remain elusive, Cx's are generally considered to mediate cell-to-cell communication by forming gap junction channels. We show here that unlike rat Cx26, human and sheep Cx26 form voltage-gated hemichannels when expressed in oocytes and Neuro2A cells. A single evolutionary amino acidic change at position 159 of the rodent protein, the replacement of aspartic acid with asparagine in the human and sheep proteins, accounts for this species specificity. At the resting potential and in normal millimolar extracellular calcium, open human Cx26 hemichannels can be detected both electrophysiologically and by dye uptake, although they did not affect cell viability. These hemichannels opened at approximately -50 mV and their activation increased by depolarization until they inactivate at positive membrane potentials. Single-channel analysis revealed that activation and inactivation involved two distinct voltage gating mechanisms and that the fully open hemichannel displays a conductance twice that of the intercellular channel. The existence of a hemichannel that opens under physiological control of the membrane potential may have important implications for the normal and pathological activity of Cx26 in humans, particularly with respect to hearing and the epidermis.

Severe neuropathy with leaky connexin32 hemichannels.

X-linked Charcot-Marie-Tooth disease is one of a set of diseases caused by mutations in gap junction proteins called connexins. We identified a connexin32 missense mutation (F235C) in a girl with unusually severe neuropathy. The localization and trafficking of the mutant protein in cell culture was normal, but electrophysiological studies showed that the mutation caused abnormal hemichannel opening, with excessive permeability of the plasma membrane and decreased cell survival. Abnormal leakiness of connexin hemichannels is likely a mechanism of cellular toxicity in this and perhaps other diseases caused by connexin mutations.

Molecular basis of calcium regulation in connexin-32 hemichannels.

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca2+ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca2+ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of approximately 90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels ( approximately 18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca2+ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca2+ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca2+ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.