A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger

Abstract

Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of beta-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten beta-sheets and two short alpha-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.

Relevance of the protein macrodipole in the membrane-binding process. Interactions of fatty-acid binding proteins with cationic lipid membranes

“…The bands at 1662 and 1684 cm -1 , contributed to 13% of the total area. This is in agreement with previous results [ 47 ]. The FTIR spectrum remained unchanged when the protein was mixed with zwitterionic membranes (not shown).…”

Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+exchanger

“…The most likely candidates are (i) palmitic acid, which is abundant in squid axons (Zlatanos et al, 2006), (ii) PtdIns(4,5)P 2 , which regulates the cardiac exchanger, and (iii) retinoic acid (RA), as suggested by the fact that ReP1-NCXSQ has a sequence profile that places it next to the CRABP family. In this regard, it is interesting that ReP1-NCXSQ binds polyphosphoinositides and phosphatidic acid but does not seem to bind other membrane components such as cholesterol, sphingomyelin, phosphoserine, phosphoethanolamide or PtdIns (Berberiá n et al, 2009).…”

“…The expression and purification of His-tagged native and mutant ReP1-NCXSQ were performed as described previously (Berberiá n et al, 2009). Membrane vesicles were obtained from the optic nerves of two squid species (S. sepioidea and L. pealei), subjected to homogenization and differential centrifugation as described elsewhere (Beaugé et al, 1996;DiPolo et al, 1997) and loaded with 300 mM NaCl, 0.1 mM Tris-EDTA, 30 mM Tris-MOPS (pH 7.3 at 293 K).…”

“…strongly suggests that Tyr128 is directly involved in palmitic acid transport. In summary, even when the actual details of the interaction of ReP1-NCXSQ with the exchanger remain elusive (Berberiá n et al, 2009), the LC-HRMS strongly suggests that ReP1-NCXSQ transports palmitic acid and that Tyr128 is essential for this function.…”

“…This protein was observed in several squid species, including Loligo pealei, Doryteuthis plei and Sepiotheutis sepioidea. Originally identified as a lipocalin (Berberiá n et al, 2009), this protein is actually placed at the frontier between the CRABP and FABP families (see x3). FABPs and CRABPs are members of the same superfamily.…”