INTERCELLULAR COMMUNICATION IN THE LENS

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DESCRIPTION: Specific Aim #1: The PI will compare single channel properties of Cx46 and Cx56 hemichannels and their regulation by Ca++, protons and phosphorylation. Goals will be to see if Cx46 and Cx50 (or Cx56 and Cx45.6, the chicken counterparts) can form heteromeric channels with novel functional properties. The PI will mutate connexins to locate Ca++ binding sites responsible for channel closure and thus localize amino acids in or near the pore of the channel. She will determine the role of the C-terminus in pH regulation of junctional channels by engineering mutants of lens fiber connexins which lack portions of the cytoplasmic C-terminus. The applicant has the unique advantage in her experimental system of looking over time at single channels and thus being able to determine dissociation constants for Ca++ interaction with the hemichannels. She will study the effects of extracellular and intracellular Ca++ on the junctional channels. With the heteromeric experiments, PI will co-express two connexins and look for channels with properties unique from those predicted from parents alone. This is a feasible approach since other connexins have been mixed and have resulted in unique physiologies in their intercellular channels. The PI will also study differences in Ca++ sensitivity of Cx46 and Cx56 hemichannels. She will construct chimeras to see which result in a switch of sensitivity. The PI will look at changes in pH sensitivity of Cx56 truncation mutants. Specific Aim #2: The PI will characterize the functional properties of lens channels transfected into N2A or HeLa cells. Questions to be addressed are: to what extent can the properties of hemichannels be correlated with intercellular channels? How do 46 and 50 differ? Do 46 and 50 make heteromeric connexons? What are the effects of stimulating phosphorylation on channel properties? The applicant has arranged a collaboration with Dr. Eric Beyer. Beyer already has Cx50 N2A transfectants. These studies will be done with double whole-cell patch clamp, focusing on some of the mutant connexins developed in the oocyte system. The PI has presented some preliminary data showing that she can obtain DWCPC data.