Cx46 and Cx50 hemichannels: Unique properties and potential roles in lens physiology and gap junction formation

Gap junction channels between lens fiber cells play a critical role in maintaining, regulating, and directing a distinctive pattern of current flow through and around vertebrate lenses. This current flow may drive a similar pattern of water flow, thereby establishing an internal circulation providing for nutrient delivery and waste removal in the avascular lens tissue. The connexins expressed by lens fiber cells can form functional hemi-gap-junction channels in single plasma membranes. The goals of this dissertation were to compare and contrast the properties of cx46 and cx50 hemichannels and their roles in regulating conductance and assembly of gap junctions. Studies were carried out on single and paired Xenopus laevis using single and dual Two-Electrode-Voltage-Clamp techniques.;Cx50 hemichannel conductance decreases in response to voltages of either polarity. An asymmetry in the voltage response was used to show that the voltage-dependent behavior of cx50 gap junction channels is predicted by the voltage-dependent behavior of cx50 hemichannels at positive potentials. Both cx46 and cx50 hemichannels were equally sensitive to [Ca2+]out, but cx50 hemichannels were dramatically more sensitive to pHout. The pH sensitivity of cx50 hemichannels was explored by determining the accessibility of presumptive pore-lining histidine residues to histidine-modifying reagents and by generating histidine mutations. The data reveal how hemichannels are regulated and their possible role in cataract.;The process of gap junction formation was examined by first determining the quantitative relationship between hemichannel conductance and gap junctional conductance. During these studies, I discovered that activating hemichannel current beyond a threshold level stimulated the rapid appearance of junctional conductance. Neither structural changes induced by voltage or [Ca2+ ]out nor intracellular changes induced by opening hemichannels could account for experimental results. Instead, data were consistent with the hypothesis that hemichannel current induces an iso-osmotic decrease of interstitial volume to bring membranes closer together so that opposing hemichannels can bind to form gap junction channels. The initial rate and extent of stimulated junctional conductance were proportional to the product of hemichannel conductances, the first direct evidence that functional hemichannels are the immediate precursors to gap junction channels. Junction assembly followed bimolecular reaction kinetics with a calcium-sensitive rate-limiting step, possibly involving carbohydrate interactions between endogenously expressed surface factors.