The Crystal Structure of the Primary Ca2+ Sensor of the Na+/Ca2+ Exchanger Reveals a Novel Ca2+ Binding Motif

摘要

The Na+/Ca2+ exchanger is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is governed by Ca2+, and the primary Ca2+ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. Here, we present a crystal structure of CBD1 at 2.5Å resolution, which reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing. The Na+/Ca2+ exchanger is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is governed by Ca2+, and the primary Ca2+ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. Here, we present a crystal structure of CBD1 at 2.5Å resolution, which reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing. Rapid fluxes of Ca2+ across the sarcolemmal membrane are an important component of cardiac excitation-contraction coupling. Ca2+ influx mediated by voltage-dependent Ca2+ channels initiates contractions, while Ca2+ efflux is dominated by the Na+/Ca2+ exchanger (1Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer, Boston2001: 133-160Google Scholar). Thus, the Na+/Ca2+ exchanger is an important component of regulation of cardiac contractility. Under most physiological conditions, the exchanger uses the energy stored in the inwardly directed Na+ gradient to catalyze the extrusion of Ca2+ from the cell with a stoichiometry of 3 Na+ for 1 Ca2+. Activity of the Na+/Ca2+ exchanger is modulated by the binding of Ca2+ to a high affinity regulatory site on an intracellular portion of the protein. Regulatory Ca2+ is not transported but potently activates exchange activity. Recent evidence suggests that Ca2+ may bind to and dissociate from its regulatory site during the rapid Ca2+ fluctuations that occur during a cardiac contraction cycle (2Ottolia M. Philipson K.D. John S. Biophys. J. 2004; 87: 899-906Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The Na+/Ca2+ exchanger protein is predicted to consist of nine transmembrane segments and a large intracellular loop (3Philipson K.D. Nicoll D.A. Ottolia M. Quednau B.D. Reuter H. John S. Qiu Z. Ann. N. Y. Acad. Sci. 2002; 976: 1-10Crossref PubMed Scopus (103) Google Scholar, 4Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The transmembrane segments translocate ions across the membrane, and the intracellular loop is largely responsible for regulation of activity. We have previously identified a region of the intracellular loop of the exchanger (amino acids 371–508) that binds Ca2+ with high affinity and mediates activation of exchange activity by Ca2+ (5Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar, 6Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). This segment comprises the first of two tandem Calx-β domains (7Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar). Mutational analysis identified two groups of three aspartate residues within the first Calx-β dom