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

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 mu M, Hill coefficient n=12), but not by the gap junction channel blocker 18 alpha-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 18 alpha-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.