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

Abstract

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 2 mM to 20 nM 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 290 mOsm 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=38 μM, 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.