The Cyclic AMP Signaling Pathway and Direct PKA Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function

CANTERO MARÍA DEL ROCIO; VELÁZQUEZ IRINA FLORENCIA; ANDREW J. STREETS; ALBERT C.M. ONG ANDREW J. STREETS BUSCAR UNIV; CANTIELLO HORACIO F

Los Angeles, California

Congreso; 60 TH Annual Meeting Biophysical Society; 2016

Biophysical Society

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent diseasecaused by mutations in the genes encoding polycystins 1 and 2, which form acomplex where the effect systems lie in polycystin-2 (PC2). PC2 is a TRP-type,Ca2þ-permeable non-selective cation channel that plays an important role inCa2þ signaling in renal and non-renal cells. The effect(s) of the cAMP pathwayand kinase mediated phosphorylation of PC2 seem to be relevant to PC2 traf-ficking and its interaction with polycystin-1. However, the role of PC2 phosphorylationin channel function is still poorly defined. Here we reconstitutedapical membranes of term human syncytiotrophoblast (hST), containing endogenousPC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition ofthe catalytic subunit of PKA increased by 566% the spontaneous PC2hst a channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase.Either stimulation was inhibited by addition of alkaline phosphatase,which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuvermodified the single channel conductance but instead increased channelmean open time. PKA directly phosphorylated PC2, which increased the meanopen time but not the single channel conductance of the channel. PKA phosphorylationdid not modify either R742X truncated or S829A-mutant PC2ivchannel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channelregulation centers on a single residue Serine 829, in the carboxy terminusthat seems to be a gating mechanism of channel activity. The fact that thecAMP/PKA pathway participates in the regulation of PC2 channel functionis crucial for the understanding of renal physiology and the pathogenesis ofADPKD