Conductive and kinetic properties of connexin45 hemichannels expressed in transfected HeLa cells

Human HeLa cells transfected with mouse connexin Cx45 were used to examine the conductive and kinetic properties of Cx45 hemichannels. The experiments were carried out on single cells using a voltage-clamp method. Lowering the [Ca2+](o) revealed an extra current. Its sensitivity to extracellular Ca2+ and gap junction channel blockers (18alpha-glycyrrhetinic acid, palmitoleic acid, heptanol), and its absence in non-transfected HeLa cells suggested that it is carried by Cx45 hemichannels. The conductive and kinetic properties of this current, I-hc, were determined adopting a biphasic pulse protocol. I-hc activated at positive V-m and deactivated partially at negative V-m. The analysis of the instantaneous I-hc yielded a linear function g(hc,inst) = f(V-m) with a hint of a negative slope (g(hc,inst): instantaneous conductance). The analysis of the steady-state I-hc revealed a sigmoidal function g(hc,ss) = f(V-m) best described with the Boltzmann equation: V-m,V-0 = -1.08 mV, g(hc,min) = 0.08 (g(hc,ss): steady-state conductance; V-m,V-0: V-m at which g(hc,ss) is half maximally activated; g(hc,min): minimal conductance; major charge carriers: K+ and Cl-). The g(hc) was minimal at negative V-m and maximal at positive V-m. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. I-hc deactivated exponentially with time, giving rise to single time constants, tau(d). The function tau(d) = f(V-m) was exponential and increased with positive V-m (tau(d) = 7.6 s at V-m = 0 mV). The activation of I-hc followed the sum of two exponentials giving rise to the time constants, tau(a1) and tau(a2). The function tau(a1) = f(V-m) and tau(a2) = f(V-m) were bell-shaped and yielded a maximum of congruent to 0.6 s at V-m congruent to -20 mV and congruent to 4.9 s at V-m = 15 mV, respectively. Neither tau(a1) = f(V-m) nor tau(a2) = f(V-m) coincided with tau(d) = f(V-m). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes.