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Pflügers Archiv

, Volume 452, Issue 4, pp 396–406 | Cite as

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

  • Patrick Bader
  • Robert WeingartEmail author
Cell and Molecular Physiology

Abstract

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 Clchannel blocker (binding constant K d=38 μM, Hill coefficient n=12), but not by the gap junction channel blocker 18α-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 18α-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.

Keywords

Gap junction hemichannel Connexin45 Volume-regulated anion channel Electrophysiology Pharmacology 

Notes

Acknowledgements

We thank D. Lüthi for technical assistance and B. Nilius, University of Leuven, Belgium, for suggesting the use of mibefradil. Transfectants were provided by K. Willecke, Institute of Genetics, University of Bonn, Germany. Supported by the Swiss National Science Foundation (31-55297.98, 31-67230.01).

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Department of PhysiologyUniversity of BernBernSwitzerland

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