A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1

Abstract

Gq protein-coupled receptor stimulation increases sarcolemmal Na+/H+ exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A1 receptor stimulation inhibits this response through a Gi protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A1 receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the α1-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A c) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate Gi proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A c and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2Ac dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A1 receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve Gi protein-mediated translocation of PP2Ac and NHE1 dephosphorylation. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.