Purchase this article with an account.
X. Li, C. K. Mitchell, R. Heidelberger, J. O'Brien; The Interaction of Calmodulin With Connexin 35 and Its Effects on Gap Junction Coupling. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3051. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Nearly all gap junctions are uncoupled by high intracellular calcium although the physiological significance of this is unknown. Previously, we have reported an interaction between neuronal gap junction protein connexin35 (Cx35/36) and calmodulin (CaM) having a low micromolar K1/2 for Ca2+. Given the widespread occurrence of Cx35/36 in mixed synapses containing Ca2+ permeable NMDA receptors, we hypothesized that this interaction may regulate gap junctional coupling during normal synaptic signaling. In this study, we examine the CaM binding sites of Cx35 and evaluate whether CaM binding can regulate gap junction coupling.
GST fusion proteins containing the C-terminal domain of Cx35 were expressed in bacteria and purified. A series of mutants (KI261EA, I267A, R268A, VQ270AA and I277A) were generated to disrupt elements of putative CaM binding motifs. Interactions between CaM and the Cx35 proteins were quantitatively measured by surface-plasma resonance. Mutants that reduced CaM binding were introduced into full-length Cx35, and expressed in stably-transfected Hela cells. Gap junctional coupling was measured by scrape-loading in the presence or absence of ionomycin to elevated intracellular Ca2+.
CaM binding to the wildtype Cx35 C-terminus had an affinity about 1.02 ± 0.16 (µM) (n=5) and K1/2 for Ca2+ between 8 to10 µM. K261 and I262 were essential for this interaction, since the amount of CaM bound to KI261EA mutant decreased to 7% compared to WT. The binding affinity for I267A and R268A were decreased to 6.05 ± 0.6 (µM) and 2.66 ± 0.25 (µM), respectively. The affinity of I277A and VQ270AA mutants were not significantly different than WT, suggesting that the site does not contain an IQ-like motif.
The C-terminal region of Cx35 is the major interacting site for CaM and two residues within it are especially critical. CaM-Cx35 interaction may be the mechanism for calcium-regulated gap junction communication.
This PDF is available to Subscribers Only