Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation.

Arjan Pieter Quist,Seung Keun Rhee,R Lal,H Lin

doi:10.1083/jcb.148.5.1063

Abstract

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to </=1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and beta-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation.

Highlights

Gap junctions, as specialized plasma membrane structures, allow the diffusion driven transfer of small cytoplasmic molecules and ions between interconnected cells which, in turn, influence cellular growth and differentiation, metabolic homeostasis, and the synchronization of electrical activity (Bennett et al, 1991; Kumar and Gilula, 1996)
In the present study, using multimodal atomic force microscopy (AFM1; for review, see Lal and John, 1994; Lal and Proksch, 1997), confocal laser immunofluorescence imaging, and fluorescent dye uptake assay, we examined the change in cell volume due to the opening of hemichannels in a variety of cells expressing or not expressing connexins
We used a combined light fluorescence and atomic force microscope (AFM) to examine the change in cell volume and associated mechanical properties resulting from the modulation of hemichannel activity

Summary

Introduction

As specialized plasma membrane structures, allow the diffusion driven transfer of small cytoplasmic molecules and ions between interconnected cells which, in turn, influence cellular growth and differentiation, metabolic homeostasis, and the synchronization of electrical activity (Bennett et al, 1991; Kumar and Gilula, 1996). Hemichannels consisting of multimeric gap-junctional proteins (connexins) are preassembled in cytoplasmic membranes and transported to the cell plasma membrane by vesicle trafficking (Musil and Goodenough, 1993; Rahman et al, 1993; Lampe, 1994; Laird, 1996; Falk et al, 1997). Some of these hemichannels interact with their counterparts from the apposing cells to form whole gap junction. Gap-junctional hemichannels could be opened in a Ca2ϩ-free medium (Pfahnl and Dahl, 1999), it is unknown whether the normal physiological fluctuations in the extracellular [Ca2ϩ] modulate hemichannel activity and whether such altered hemichannel activity serves any physiological functions

Methods

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Conclusion