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A.M. DeRosa, R. Mui, M. Srinivas, T.W. White; Functional Characterization of a Naturally Occurring Cx50 Truncation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1854.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Connexin proteins undergo proteolytic cleavage of their c–termini during lens fiber differentiation. While the functional significance of this is not fully understood, connexin cleavage has been correlated with changes in both gap junctional organization and channel gating, although different results with regard to gating were obtained by different laboratories. To further evaluate the functional consequences of connexin cleavage, we have generated and characterized a c–terminally truncated Cx50 protein. Methods: Mouse Cx50 was truncated at amino acid 290 and expressed in paired Xenopus oocytes or N2A cells. Protein expression was confirmed by western blotting. Gap junctional conductance and voltage dependent gating were measured using the dual whole–cell voltage clamp technique. pH dependent gating was determined by perfusing cells with media saturated with 100% CO2. Results: Truncated Cx50 protein was expressed and formed channels with macroscopic conductance levels similar to those of the wildtype connexin. The voltage dependant gating of truncated Cx50 was comparable to the full length protein. The unitary conductance of truncated Cx50 was ∼215 pS in 130 mM CsCl, a value very close to that reported for full length Cx50 (∼220 pS) and showed voltage dependant open probability (Po) similar to wildtype channels. Po decreased with increasing transjunctional voltage. Finally, the conductance of both truncated and wildtype Cx50 were reversibly reduced by cytoplasmic acidification. Conclusions: We found that c–terminal truncation of Cx50 did not significantly alter any of the functional properties analyzed. Our results suggest that connexin cleavage may be more important for the structural organization of gap junctions rather than regulation of the intrinsic channel properties.
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