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Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels

Bader, Patrick ; Weingart, Robert

In: Pflügers Archiv, 2006, vol. 452, no. 4, p. 396-406

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    Titre
    • Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels
    Auteur
    • Bader, Patrick. Department of Physiology, University of Bern, Bühlplatz 5, CH-3012, Bern, Switzerland
    • Weingart, Robert. Department of Physiology, University of Bern, Bühlplatz 5, CH-3012, Bern, Switzerland
    Type de document
    • Postprint
    Identifiant OAI-PMH
    • oai:doc.rero.ch:321853
    Summary
    Human HeLa cells transfected with mouse connexin45 were used to explore the experimental conditions suitable to measure currents carried by gap junction hemichannels. Experiments were performed with a voltage-clamp technique and whole-cell recording. Lowering [Ca2+]o from 2mM to 20nM evoked an extra current, I m, putatively carried by Cx45 hemichannels. However, the variability of I m (size, voltage sensitivity, kinetics) suggested the involvement of other channels. The finding that growth medium in the incubator increased the osmolarity with time implied that volume-regulated anion channels (VRAC) may participate. This assumption was reinforced by the following observations. On the one hand, keeping [Ca2+]o normal while the osmolarity of the extracellular solution was reduced from 310 to 290mOsm yielded a current characteristic of VRAC; I VRAC activated/deactivated at negative/positive voltage, giving rise to the conductance functions g VRAC,inst=f(V m) (inst: instantaneous; V m: membrane potential) and g VRAC,ss=f(V m) (ss: steady state). Moreover, it was reversibly inhibited by mibefradil, a Cl-channel blocker (binding constant K d=38μM, Hill coefficient n=12), but not by the gap junction channel blocker 18α-glycyrrhetinic acid. On the other hand, minimizing the osmotic imbalance while [Ca2+]o was reduced led to a current typical for Cx45 hemichannels; I hc activated/deactivated at positive/negative voltage. Furthermore, it was reversibly inhibited by 18α-glycyrrhetinic acid or palmitoleic acid, but not by mibefradil. Computations based on g VRAC,ss=f(V m) and g hc,ss=f(V m) indicated that the concomitant operation of both currents results in a bell-shaped conductance-voltage relationship. The functional implications of the data presented are discussed. Conceivably, VRAC and hemichannels are involved in a common signaling pathway