Conductive and Kinetic Properties of Connexin45 Hemichannels Expressed in Transfected HeLa Cells

Abstract

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 (18α-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,0 = −1.08 mV, g _hc,min = 0.08 (g _hc,ss: steady-state conductance; V _{m, 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, τ_d. The function τ_d = f(V _m) was exponential and increased with positive V _m (τ_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, τ_a1 and τ_a2. The function τ_a1 = f(V _m) and τ_a2 = f(V _m) were bell-shaped and yielded a maximum of ≅ 0.6 s at V _m ≅ −20 mV and ≅ 4.9 s at V _m ≅ 15 mV, respectively. Neither τ_a1 = f(V _m) nor τ_a2 = f(V _m) coincided with τ_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.