Conductive and Kinetic Properties of Connexin45 Hemichannels Expressed in Transfected HeLa Cells |
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Authors: | P?Bader Email author" target="_blank">R?WeingartEmail author |
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Institution: | (1) Department of Physiology, University of Bern, Bern, CH-3012, Switzerland |
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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, τd. The function τd = f(V
m) was exponential and increased with positive V
m (τd = 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, τa1 and τa2. The function τa1 = f(V
m) and τa2 = 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 τa1 = f(V
m) nor τa2 = f(V
m) coincided with τd = 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. |
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Keywords: | Gap junction hemichannel Connexin45 Conductance Kinetics Electrophysiology Heart |
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