Effect of Ca²⁺ on Protein Kinase A‐Mediated Phosphorylation of a Specific Serine Residue in an Expressed Peptide Containing the Ca²⁺‐Regulatory Domain of Scallop Muscle Na⁺/Ca²⁺ Exchanger

Abstract

Sequencing of the scallop muscle Na+/Ca2+ exchanger revealed three consensus sequences for phosphorylation by PK-A in the large cytoplasmic loop (R(363)KLTG, R(379)RASV, and R(618)RGSV). Site-directed mutagenesis of the expressed Glu(384)-Ser(713) segment of the f loop identified Ser(621)as a residue phosphorylated by PK-A. The R(618)RGSV sequence is located at the junction of the mutually exclusive exon and exon 9, a site where many alternatively spliced variants of vertebrate NCX1 and NCX3 are generated. Phosphorylation of Ser(621) by PK-A in the isolated Glu(384)-Ser(713) peptide was blocked under conditions where Ca2+ was bound.

Pharmacological Characterization of the Newly Synthesized 5-Amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide Hydrochloride (BED) as a Potent NCX3 Inhibitor That Worsens Anoxic Injury in Cortical Neurons, Organotypic Hippocampal Cultures, and Ischemic Brain

“…Interestingly, the substitution on the amino group with an acetamide group abolishes the effect on NCX (data not shown), thus showing that the amino group plays an important role in the inhibitory effect exerted by the newly synthesized drug. The use of deletion mutagenesis showed that the intracellular f-loop of NCX3, which is involved in the activity regulation of the antiporter, , is not involved in BED inhibitory action. In contrast, the use of NCX1/NCX3 chimeras revealed that both the α 1 and α 2 repeats were mainly involved in BED inhibitory action.…”

Cell Calcium Extrusion Systems and their Role in Epileptogenesis~!2009-05-14~!2010-01-06~!2010-04-21~!

“…Other protein-regulating factors include pH, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), ATP, phosphorylation [71]; as well as the exchanger inhibitor peptide (XIP), which completely inhibits activity when interacting with the N-terminal segment of the large intracellular loop [69]. There are a number of proteins that associate with the NCX1 in the intracellular loop that regulate exchanger transport function, such as protein kinases A (PKA) and C (PKC) [72][73][74]. It has been also found that phosphatases PP1 and 2A are associated with NCX1.…”

Cardiac-Specific Overexpression of NCX1.1-Xip Mutant Causes Dilated Cardiomyopathy in Mice

“…Overexpression, Purification and Functional Characterization of the Human Serotonin Transporter Hidehito Takayama 1,2 , Shigetoshi Sugio 1,2 . 1 Mitsubishi Chemical Corporation, Yokohama, Japan, 2 Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Japan. The human serotonin transporter (SERT) regulates the concentration of serotonin, a neurotransmitter, in the synaptic cleft through the reuptake of serotonin.…”

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Site directed mutagenesis has shown that Ser 621 in the CBD2 sub-domain of the Ca 2þ -regulatory domain of scallop muscle Na þ -Ca 2þ exchanger (NCX-SCA) is a substrate for cAMP-PK (PK-A) in the native membrane -bound enzyme [1,2], and under the same conditions Na þ -driven 45 Ca 2þ uptake is stimulated [3]. Examination of the amino acid sequence of NCX-SCA (AY567834, GenBank) shows a consensus sequence for AMP-PK in CBD2 in the Ca 2þ -regulatory domain, with Ser 621 as the target residue.

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“…Fukuoka University School of Medicine, Fukuoka, Japan, 2 Toho University School of Medicine, Tokyo, Japan, 3 Chiba University Graduate School of Medicine, Chiba, Japan, 4 Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, 5 National Cardiovascular Center, Osaka, Japan. The Na þ /Ca 2þ exchanger (NCX1.1) plays the primary role in Ca 2þ extrusion from cardiac myocytes during diastole.…”