Identification and Characterization Of Gap Junctional Hemichannels From Dissociated Mouse Lens Epithelial And Newly Differentiating Fiber Cells

Abstract

Purpose:: To identify and characterize the macroscopic and single properties of connexin hemichannels in lens epithelial cells and differentiating fiber cells isolated from wild-type mice.

Methods:: The whole cell patch clamp technique was used to study membrane currents in dissociated lens epithelial cells and newly differentiating fiber cells from 4-8 day mouse lenses maintained in divalent cation-free Ringer solution. To block potassium currents, cesium was used as the main cation in the pipette solution.

Results:: Under these conditions, the primary current in the newly differentiating fiber cells was a multichannel current composed of large conductance channels. This current was partially closed at a holding potential of -60 mV, activated on depolarization, and had a reversal potential of ~ 0 mV. Application of 2 mM [Ca²⁺]_o slowed the time course of activation, accelerated the time course of deactivation, shifted the threshold of activation to ~+10 mV and significantly reduced the size of the current at positive potentials. These properties resembled those of Cx46 hemichannel currents expressed in Xenopus oocytes. Multichannel currents composed of large conductance channels were also frequently seen in the epithelial cells. In some cells, only a few channels were observed. These channels typically opened on depolarization and closed on hyperpolarization to -60 mV. They had a single channel conductance (γ₀ = 265-531 pS) that fell into the same range as Cx46 and Cx50 hemichannels.

Conclusions:: These results verify the existence, at significant density, of connexin hemichannels in dissociated epithelial and newly differentiating fiber cells.

Keywords: gap junctions/coupling • cataract • ion channels