Functional characterization of a naturally occurring Cx50 truncation.
OBJECTIVE
Lens connexins undergo proteolytic cleavage of their C termini during fiber maturation. Although the functional significance of this is unknown, cleavage has been correlated with changes in channel-gating properties. This study evaluates the functional consequences of this endogenous truncation by characterizing the properties of a C-terminal truncated Cx50 protein.


METHODS
Murine and human Cx50 were truncated at amino acids 290 and 294, respectively, before expression in paired Xenopus oocytes or mammalian cells. Protein expression was evaluated by immunocytochemistry. Dual whole-cell voltage clamp techniques were used to analyze macroscopic and single-channel conductance, voltage-gating properties, and kinetics; pH gating sensitivity was measured by superfusion with 100% CO2-saturated media.


RESULTS
Cx50tr290 channels exhibited an 86% to 89% reduction in mean macroscopic conductance compared with full-length Cx50. Heterotypic channels formed functional gap junctions, displayed an intermediate level of coupling, and exhibited unaltered voltage-gating properties. C-terminal truncation did not alter single-channel gating characteristics or unitary conductance. Interestingly, truncated and full-length Cx50 channel conductances were reversibly blocked by cytoplasmic acidification.


CONCLUSIONS
C-terminal truncation of Cx50 did not inhibit the formation of homotypic or heterotypic channels. However, a significant decrease in conductance was observed for truncated channels, a phenomenon independent of alterations in voltage-gating sensitivity, kinetics, or chemical gating. These results provide a plausible explanation for the 50% decrease in junctional coupling observed during lens fiber maturation.
DeRosa
Adam M
AM
Graduate Program in Genetics, State University of New York, Stony Brook, NY 11794-8661, USA.
Mui
Rickie
R
Srinivas
Miduturu
M
White
Thomas W
TW
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D017630:Connexins / Q000502:physiology*; D004594:Electrophysiology; D005136:Eye Proteins / Q000502:physiology*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology*; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D014162:Transfection; D014982:Xenopus laevis; D000099264:Connexin 50
D017630:Connexins; D005136:Eye Proteins; D007473:Ion Channels; D000099264:Connexin 50