Abstract
Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.
Highlights
Gap junctions comprise the intercellular channels that mediate the cell-to-cell transfer of small molecules, including metabolites, second messengers, and ions, in mammalian cells [1]
Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca2؉-dependent manner, providing a molecular basis for the well characterized Ca2؉-dependent inhibition of Cx43-containing gap junctions
Cell-to-cell transfer of the fluorescent dye AlexaFluor594 was half-maximally inhibited at ϳ300 nM [Ca2ϩ]i in lens cell cultures, and this inhibition was prevented by preincubation of these cultures with CaM antagonists [6, 7], consistent with earlier reports that elevated [Ca2ϩ]i increased internal electrical resistance in the lens that was prevented by preincubation with CaM antagonists [6]
Summary
Connexin; aa, amino acids; CaM, calmodulin; Mops, 4-morpholinepropanesulfonic acid; TFE, trifluoroethanol; WT, wild type. Duffy et al [11] were unable to detect any interaction of CaM with a peptide spanning the first 21 amino acids of the N terminus Both groups, failed to detect the binding of CaM to peptides derived from the C-terminal tail (aa 314 –325, 336 –350, and 346 –360) or the intracellular loop (aa 95–114, 123–136, and 119 –144). These results suggest that the interaction of CaM with Cx43 might occur via other regions of the intracellular loop. Our findings strongly suggest that Ca2ϩ effects the inhibition of Cx43 via a Ca2ϩ-dependent interaction between the CaM and residues 136 –158 of this ubiquitous gap junction protein
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