Lens cell-to-cell channel protein: II. Conformational change in the presence of calmodulin.
Lens fibers are coupled by communicating junctions, clusters of cell-to-cell channels composed of a 28-kD intrinsic membrane protein (MIP26). Evidence suggests that these and other cell-to-cell channels may close as a result of protein conformational change induced by activated calmodulin. To test the validity of this hypothesis, we have measured the intrinsic fluorescence emission and far-ultraviolet circular dichroism of the isolated components MIP26, calmodulin, and the MIP26-calmodulin complex, both in the absence and presence of Ca++, an uncoupling agent. MIP26 shows no change in either fluorescence emission (primarily tryptophan and a measure of aromatic constitutivity) or in its circular dichroism spectrum. Calmodulin exhibits a 32% increase in fluorescence emission intensity with constant emission wavelength, entirely tyrosine, and a 44% increase in alpha-helicity, changes previously described. The MIP26-calmodulin complex, on the other hand, displays fluorescence emission and circular dichroism spectra which are slightly different from the sum of the two single components, but shows marked differences in both spectra upon Ca++ addition. This indicates a change in conformation in one or both of the two components. Spectral changes include a 5-nm blue-shift, a 50% increase in tyrosine fluorescence emission, a 25% decrease in tryptophan fluorescence emission, and a 5% increase in the alpha-helicity of the complex. These changes also occur about an isosbestic point and are fully reversible. These data provide additional evidence that activated calmodulin may modulate gating of cell-to-cell channels by affecting channel protein.
D000818:Animals; D020346:Aquaporins; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D002942:Circular Dichroism; D005136:Eye Proteins / Q000378:metabolism*; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D007700:Kinetics; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D020346:Aquaporins; D002147:Calmodulin; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C030328:aquaporin 0