Gating by Voltage and Ca2+ in Human Connexin (cx26) Hemichannels

Abstract

Opening of connexin hemichannels permits the release of small metabolites, such as ATP and glutamate, which play an important autocrine/paracrine signaling in a variety of cell types. The recently solved crystal structure of the Cx26 gap junction channel allows us to explore in greater detail the relationship between the structure and function of both, hemichannels and gap junction channels. Here, we begin by revisiting the activation mechanisms of human Cx26 (hCx26) hemichannels by voltage and Ca2+. Using the two electrode oocyte voltage-clamp technique, we found that depolarization up to +60 mV induced activation of hemichannel currents, and repolarization produced large tail-currents with slow deactivation (τ ∼10 s). Interestingly, the magnitudes of the tail-currents were dependent on the lengths of the depolarizating pulses rather than the magnitudes of the currents activated during the pulses. Strikingly, cell-attached single channel recordings showed that depolarizating pulses (≤40 mV) stimulated only infrequent and brief openings of hCx26 hemichannels. However, the repolarizating pulses induced opening of single hemichannel currents (likely corresponding to the tail currents) with smaller conductance and longer mean open times. In addition, we found that low Ca2+ (500 μM) increased macroscopic hemichannel currents at positive potentials and slowed deactivation of the tail currents. We are currently performing single channel recordings to elucidate how Ca2+ modulates channel activation and deactivation kinetics in response to depolarizing and repolarizing voltages. Our results indicate that while depolarization causes opening of some hCx26 hemichannels, it mostly shifts the hemichannels to a non-conductive state that will likely open after repolarization; and that Ca2+ ions may play a role by regulating the energetics of these transitions. Supported by The Grass Foundation.