Activation of (Na++K+)-ATPase (NKA) regulates cardiac L-type Ca2+ channel (LTCC) function through molecular crosstalk. The mechanism underlying NKA-LTCC crosstalk remains poorly understood. We have previously shown that activation of NKA leads to phosphorylation of LTCC alpha(1) Ser(1928). Here we investigated whether LTCC beta(2) subunit is modulated by NKA activation and found that LTCC beta(2) Ser(496) is phosphorylated in response to activation of NKA. Src inhibitor PP1 and Erk1/2 inhibitor PD98059 abolish LTCC beta(2) Ser(496) phosphorylation, suggesting that NKA-mediated beta(2) Ser(496) phosphorylation is dependent of Src/Erk1/2 signaling pathway. Protein kinase G (PKG) inhibitor KT5823 failed to inhibit the phosphorylation of beta(2) Ser496, indicating that the NKA-LTCC crosstalk is independent of PKG activity. The results of nifedipine sensitive Ca-45 influx experiments suggest that phosphorylation of beta(2) Ser(496) may play a key down-regulation role in attenuating the accelerated activity of alpha(1) subunit of the channel. Ouabain does not cause a phosphorylation on beta(2) Ser(496), indicating a fundamental difference between activation and inhibition of NKA-mediated biological processes. This study provides the first evidence to demonstrate that LTCC beta(2) subunit is coupled with the movement of signals in the mechanism of activation of NKA-mediated crosstalk with LTCC. (C) 2010 Elsevier Inc. All rights reserved.