Regulation of Dye Permeability in Mechanically-Sensitive Culture Cells

Abstract

Introduction: Gap junction channels, consisting of two hemichannels in apposed cells, exist in many mammalian tissues and provide a conduit for communication between cells. When the hemichannels are not docked with others, evidence has shown that these hemichannels open in response to varying stimuli and contribute to cellular function. The gating of the hemichannels is regulated by external mechanical and ionic stimuli which result in varied changes to the hemichannel open probability.

Objective: An effort was undertaken to better understand the importance and influence of calcium on hemichannel regulation as this effect is still not well understood. This present evaluation utilized two reagents to influence hemichannel open probability. Gadolinium was utilized for its characteristic blocking of the stretch-activated channels in the plasma membrane. Thapsigargin, known to effect SERCA pumps, was utilized to empty the intracellular calcium stores and modify the cytoplasmic calcium concentration.

Methods: Through the use of dye uptake studies incorporating mechanical stimulation, in varied external calcium concentrations, the response of Cx43-HeLa cells to these reagents was evaluated.

Results: We have confirmed previous studies in that hemichannels are gated closed in response to mechanical stimulation and normal external calcium concentration, but will increase the open probability in response to mechanical stimulation and calcium free external environment. Data supporting reduction in hemichannel open probability, from Cx43-HeLa cells treated with gadolinium, are consistent with the presence of stretch-activated channels in the plasma membrane. Cx43-HeLa cells treated with thapsigargin resulted in additional opening of hemichannels above that observed from mechanical stimulation with or without the presence of calcium in the external environment.

Conclusion: These data support the finding that connexin and pannexin hemichannels are mechanically-sensitive membrane channels. Connexin hemichannels reduce the open probability in response to raising external calcium. In addition, while in normal external calcium pannexin hemichannels increased their open probability in response to increased cytoplasmic calcium after thapsigargin treatment.