14-3-3τ Promotes Surface Expression Of Cav2.2 （α1B） Ca²⁺ Channels

Aim: The surface expression of voltage-gated calcium channels(Cav) is important for their function in calcium homeostasis in the physiology of excitable cells, but whether and how the a1 pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, b and a(17)d(11) remained mysterious. The present study focuses on enucleating the role of 14-3-3 proteins in Cav2.2 channel trafficking.Contents: To elucidate the regulatory function of 14-3-3 proteins in the surface expression of Cav2.2 channels and possible mechanism of 14-3-3-mediated Cav2.2 channel trafficking.Methods: Reverse transcription-polymerase chain reaction(RT-PCR) analysis detected Cav auxiliary subunit in ts A-201 cells and mouse brains. Coimmunoprecipitation and Western Blot assessed the interaction of 14-3-3 proteins with the C-terminal fragments of Cav2.2 pore forming a1B subunit. Immunofluorescence staining and Confocal microscopy evaluated surface expressed and total Cav2.2α1B or Cav2.2α1B C terminus protein levels in ts A-201 cells cotransfected either HA-a1B or Myc-CD8a-Cav2.2 C terminus with overexpression 14-3-3 proteins or disruption the 14-3-3 binding to HA-a1B or Myc-CD8a-Cav2.2 C terminus by 14-3-3 inhibitor peptide(p SCM138). Whole-cell voltage clamp recording examined Cav2.2 current density over a wide range of test potentials in ts A-201 cells cotransfected Cav2.2α1B with overexpression 14-3-3 proteins or Cav b subunits or disruption the 14-3-3 binding to Cav2.2α1B by p SCM138 and in primary cultured hippocampal neurons transfected with p SCM138 or its inactive mutant control, p SCM174.channel in transfected ts A-201 cells in the absence of any known Cav auxiliary subunit. Coexpression of 14-3-3 antagonist construct, p SCM138, but not its inactive control, p SCM174, markedly suppressed 14-3-3-mediated Cav2.2α1B surface expression. Between the two previously identified 14-3-3 binding regions at the a1B C-terminus(CT1, aa1706-1940; CT2, aa2102-2220), only the proximal region(CT1), closer to the end of the last transmembrane domain, was retained by the ER and facilitated by 14-3-3 to traffic to plasma membrane via direct protein-protein interaction. 14-3-3/Cavb subunit co-regulated the surface expression of Cav2.2 channels in transfected ts A-201 cells and neurons. Results: 14-3-3 proteins promoted surface expression of the Cav2.2α1BConclusion: 14-3-3 proteins enhance Cav2.2 trafficking by masking the ER retention signal at its proximal C-terminal region and the surface expression of Cav2.2 could be coordinately regulated by Cav b subunit and 14-3-3.