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<td><h1><font color="#FFFFFF"><strong>Mark Hilge</strong><br>
<br>
<strong>Structural Biology<br>
<br>
NCX</strong></font> </h1>
<h3><font color="#FFFFFF"> <br>
Background</font></h3>
<p><font color="#FFFFFF">The Na+/Ca2+ exchanger, in concert with the
plasma membrane Ca2+ ATPase (PMCA) and the sarcoplasmic reticulum
Ca2+ ATPase (SERCA), removes Ca2+ ions that enter the cytosol via
L-type Ca2+ channels and/or from internal Ca2+ stores during the
action potential. Driven by the Na+ gradient generated by the Na+/K+
ATPase, the exchanger predominantly expels one Ca2+ ion for the
uptake of 3 Na+ ions [1], may however, under certain conditions,
also operate in reverse mode [2]. <br>
Structurally, NCX appears to consist of nine transmembrane a-helices
[3,4] and a large, approximately 500 residue-long cytosolic loop.
Recently, we demonstrated the presence of two, rather than the previously
assumed one Ca2+ binding domain in the cytosolic exchanger loop
[5]. We also proposed a model in which the Ca2+ binding sites of
the first Ca2+ binding domain (CBD1) are approximately 90 Å
away from the transport Ca2+ binding site in the ion-conducting
transmembrane domain. In contrast, Ca2+ binding sites of the second
Ca2+ binding domain (CBD2) are close to a predicted third domain
that shows homology with a-catenin and that we therefore designated
as catenin-like domain (CLD). <br>
Ion-transport of the exchanger is regulated by concentration-dependent
interactions of Na+ and Ca2+ ions with the cytosolic exchanger domains.
Binding of Ca2+ to CBD1 with an affinity of 140-400 nM [5-7] activates
ion-transport. Once the local intracellular Ca2+ concentration drops
below the CBD1 Ca2+ affinities, the exchanger enters a process referred
to as Ca2+-dependent or I2 inactivation [8]. Concomitantly, Na+
concentrations rising above 15 mM can lead to Na+-dependent or I1
inactivation [9]. </font></p>
<h3><font color="#FFFFFF">Ca2+ sensors</font></h3>
<p><font color="#FFFFFF">We determined the solution structures of
residues 371-509 and 501-657 of canine NCX1 that encode the two
Ca2+ sensors, CBD1 and CBD2, in NCX. While CBD1 and CBD2 look very
similar in the Ca2+ bound form, [1H,15N]-HSQC spectra of the domains
in the absence of Ca2+ suggest striking differences in their apo-structures.
Mapping shifted resonances onto the molecular structure of CBD1
shows a loss of structural integrity in the Ca2+-binding sites of
the CBD1 apo-form. Examination of the two domains reveals that only
CBD2, but not CBD1, possesses basic residues in the Ca2+ binding
sites that can form salt-bridges with surrounding acidic residues
and in this way reduce repulsion. The large conformational change
upon Ca2+-binding and a 5-10 times higher affinity for Ca2+ makes
CBD1 the primary Ca2+ sensor in NCX. The markedly different behaviour
of the two domains we visualized in a molecular movie.</font></p>
<h3><font color="#FFFFFF">Data & Reprints</font></h3>
<p><font color="#FFFFFF"><strong>Reprint</strong><br>
[5] Hilge M, Aelen J, Vuister GW. Ca2+ regulation in the Na+/Ca2+
exchanger involves two markedly different Ca2+ sensors. Mol Cell
22, 15-25 (2006).<br>
DOWNLOAD CBDs.pdf 828kB</font></p>
<p><font color="#FFFFFF"><strong>Coordinates & Chemical shifts</strong><br>
The coordinates and all experimental data (chemical shifts and constraints)
of the Ca2+ bound CBD1 and CBD2 ensembles have been deposited with
the Protein Data Bank (accession codes 2FWS and 2FWU, respectively).</font></p>
<p><font color="#FFFFFF"><strong>References</strong><br>
[1] Kang TM, Hilgemann DW (2004) Nature 427:544-8.<br>
[2] Kohmoto O, Levi AJ, Bridge JH (1994) Circulation Research 74:550-554.<br>
[3] Iwamoto T, Nakamura TY, Pan Y, Uehara A, Imanaga I, Shigekawa
M (1999) FEBS Lett 446:264-8.<br>
[4] Nicoll DA, Ottolia M, Lu L, Lu Y, Philipson KD (1999) J Biol
Chem 274:910-7.<br>
[5] Hilge M, Aelen J, Vuister GW (2006) Mol Cell 22:15-25.<br>
[6] Levitsky DO, Nicoll DA, Philipson KD (1994) J Biol Chem 269:22847-52.<br>
[7] Ottolia M, Philipson KD, John S (2004) Biophys J 87:899-906.<br>
[8] Hilgemann DW, Collins A, Matsuoka S (1992) J Gen Physiol 100:933-61.<br>
[9] Hilgemann DW, Matsuoka S, Nagel GA, Collins A (1992) J Gen Physiol
100:905-32.<br>
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