Regulation Of Human Cx31 Gap Junctional Activity By Protein Kinases

1. Background1.1 Function of gap junction and regulation of gap junctional activityGap junctions are cell membrane-spanning structures consisting ofconnexins. Gap junction channels synchronize activities of adjacent cellsby regulating the direct exchange of small molecules (molecular weight＜1 kDa). Gap junction intercellular communication (GJIC) plays a keyrole in organ homeostasis of multicellular organisms. GJIC weredifferentially regulated in response to various physiological stimuliincluding transjunctional voltage and events activating protein kinases. Incontrast to transjunctional voltage that directly modulate gap junctionchannels gating, protein kinases differentially regulate GJIC at multiplecontrol points including gap junction channels establishment and singlechannel gating.1.2 Functional studies of Cx31 gap junctionConnexin31 (Cx31) is one of the homologous proteins of theconnexin family. Human Cx31 channel is relative nonselective, allowingpassage of large ions with both positive and negative charges. Like otherconnexin channels, Cx31 gap junction channels are gated by voltage.Mouse Cx31 was reported to be phosphorylated at the serine²⁶⁶ residuesand mutation of the serine²⁶⁶ to alanine results in increased turnover of Cx31 protein and decreased GJIC. Nevertheless there was no directevidence that mouse Cx31 GJIC is modulated by phosphorylation in vivo.in addition, serine²⁶⁶ is substituted by an asparagine residue in humanCx31.2. Research goalsTo explore the mechanism of Cx31 GJIC modulation, we investigatethe effect of two ubiquitous proetein kinases, PKA and PKC, on channelfunction and cellular behavior of Cx31. Our goals are to determinewhether and how PKA and PKC modulate formation and activity of Cx31gap junction. The results may contribute to our understanding of thedynamic regulation of Cx31 GJIC.4. Methodes and ResultsTo examine the effects of PKA and PKC on Cx31 GJIC, weemployed Hela cells stably expressing Cx31/myc as a model system,measured Cx31 mediated dye transfer ability after exposing cells toactivators and inhibitors of PKA and PKC. The results reveal that thePKA activator 8-Br-cAMP increase calcein coupling rate by more than2-fold compared to the untreated control with an exposure time of 3 hours.Conversely, 3 hours treatment of the PKC activator PMA dramaticallydecreased dye transfer rate to only 13ï¼…of the value of DMSO treatedcontrol. In cells treated with 8-Br-cAMP, number of fluorescent labeledgap junction plaques increased by 45ï¼…compared with the untreated control. In addition, the size of fluorescent labeled plaques is obviouslyenlarged. In contrast cells treated with PMA showed significantly changein neither number nor size of fluorescent Cx31 plaques. Immunoblottinganalysis showed that treatment with either 8-Br-cAMP or PMA did ontchange total cellular level and phosphorylation status of Cx31. BFA andnocodazole had no effect on the stimulated effects of 8-Br-cAMP,However, CytoD dramatically blocked the effects of 8-Br-cAMP.5. ConclusionsOur data suggests that PKA and PKC differentially modulate Cx31GJIC. Activation of PKA likely accelerated post-Golgi trafficking orfollowing channel assembly of the pre-existing Cx31 but not ER-Golgitransportation. Actin cytoskeleton was found to play a key role in thisprocess. Activation of PKC showed no affect on number of Cx31channels, however, it likely inhibits Cx31 channel activity. Cx31 gapjunctions is continually formed and removed. Our findings suggest thatthe dynamic process of Cx31 gap junction formation and channel gatingcan be delicately modulated by intercellular signals that activating PKAor PKC pathway.