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Connexin Type- and Protein Kinase-Specific Modulation of Gap Junction Inhibitor Potency by Phosphorylation
Date Issued |
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2022-07-16 |
Chemical modulators of gap junction intercellular communication (GJIC) are being used in cases of various communication-dependent diseases, in particular those of cardiac, neurological, and oncological origin. Recently we found that the potency of inhibition of Cx43 GJs by monoterpene a-pinene can be allosterically regulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Since Cx43 C-terminus has been shown to be phosphorylated at 21 sites by various kinases, we used dual whole-cell patch-clamp techniques and selective inhibitors to determine whether the potency of a-pinene depends on the activity of other kinases: PKC, PKA, PKG, v-Src, Akt, MAPK, casein kinase, and cyclindependent kinase. We found that in addition to CaMKII only another Ca2+- dependent kinase PKC was involved in the observed phenomenon. Further, we performed molecular docking of several known GJ inhibitors: octanol, carbenoxolone, mefloquine, flufenamic acid, glycyrrhetinic acid, and sevoflurane and found that only the potency of sevoflurane that docked to the similar site on Cx43 as a-pinene was phosphorylation-dependent. Finally, we examined whether the potency of inhibition of GJs composed of other Cx isoforms by apinene is also dependent on phosphorylation and found that non-selective inhibitor of kinases staurosporine, in contrast to Cx43 channels, did not modify the inhibitory effect of a-pinene on Cx36 and Cx45 GJs typical to neurons and cardiac cells, respectively, exhibiting high electrical activity and phosphorylation levels. Taken together, our data suggest that the observed phenomenon of allosteric modulation of GJ chemical gating is inhibitor-, protein kinase-, and Cx type-dependent.