Rat vas deferens SERCA2 is modulated by Ca(2+)/calmodulin protein kinase II-mediated phosphorylation

Ca(2+) pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca(2+)-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca(2+) (Ca(0.5) = 780 nM) and a low sensitivity to vanadate (IC(50) = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca(2+) and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca(2+) accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca(2+) and CaM, possibly via CaMKII, in a process that results in stimulation of Ca(2+) pumping activity.