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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, Ihc, were determined adopting a biphasic pulse protocol. Ihc activated at positive Vm and deactivated partially at negative Vm. The analysis of the instantaneous Ihc yielded a linear function ghc,inst = f(Vm) with a hint of a negative slope (ghc,inst: instantaneous conductance). The analysis of the steady-state Ihc revealed a sigmoidal function ghc,ss=f(Vm) best described with the Boltzmann equation: Vm,0= -1.08 mV, ghc,min=0.08 (ghc,ss: steady-state conductance; Vm,0: Vm at which ghc,ss is half-maximally activated; ghc,min: minimal conductance; major charge carriers: K+ and Cl-). The ghc was minimal at negative Vm and maximal at positive Vm. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. Ihc deactivated exponentially with time, giving rise to single time constants, taud. The function taud = f(Vm) was exponential and increased with positive Vm (taud=7.6 s at Vm=0 mV). The activation of Ihc followed the sum of two exponentials giving rise to the time constants, taua1 and taua2. The function taua1=f(Vm) and taua2 = f(Vm) were bell-shaped and yielded a maximum of congruent with 0.6 s at Vm congruent with -20 mV and congruent with 4.9 s at Vm congruent with 15 mV, respectively. Neither taua1 =f(Vm) nor taua2 = f(Vm) coincided with taud=f(Vm). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes.

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