Metabolic regulation of the squid nerve Na+/Ca2+ exchanger. Recent developments.

L. BEAUGÉ, R. DIPOLO, G. ELSO DE BERBERIAN AND A. PODYARNIK.

Lacco Ameno, Ischia Islnd, Naples

Conferencia; 6ª International Conference on Na+/Ca2+ Exchange.; 2011

In squid nerve MgATP modulation of the Na+/Ca2+ exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin super family of Lipid Binding Proteins (LBP or FABP) of 132 amino acids. We called it Regulatory Protein of Squid Nerve Sodium Calcium Exchanger (ReP1-NCXSQ, access to GenBank EU981897). ReP-1-NCXSQ was cloned, expressed and purified. Circular dichroism Far-UV and infrared spectroscopy suggest a secondary structure predominantly of beta sheets. The tertiary structure prediction provides ten beta sheets and two alpha helices, characteristic of most of LPB. Functional experiments showed that to be active ReP1-NCXSQ must be phosphorylated by MgATP through the action of a kinase present in the plasma membrane. Moreover, PO4-ReP1 NCXSQ can stimulate the exchanger in the absence of ATP. An additional crucial observation was that in proteoliposomes containing only the purified Na+/Ca2+ exchanger, PO4-ReP1-NCXSQ promotes activation; therefore, this up-regulation does not need any other requirement besides a lipid membrane and the incorporated exchanger protein. Recently, we solved the crystal structure of ReP1-NCXSQ which was as predicted: a "barrel" consisting of ten beta sheets and two alpha helices. Inside the barrel is the fatty acid coordinated by hydrogen bonds with Arg126 and Tyr128. Point mutations (Tyr20Ala, Arg126Val and Tyr128Phe) showed that neither Tyr20 nor Arg126 are necessary protein phosphorylation or activity.