Endogenous XIP Region

The cardiac sarcolemmal Na 1 -Ca 2 1 exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na 1 -dependent inactivation. This inactivation is manifested as a partial decay in outward Na 1 -Ca 2 1 exchange current after application of Na 1 to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na 1 affinities. Regulation of the exchanger by nontransported, intracellular Ca 2 1 was also modified by the XIP-region mutations. Binding of Ca 2 1 to the intracellular loop activates exchange activity and also decreases Na 1 -dependent inactivation. XIP-region mutants were all still regulated by Ca 2 1 . However, the apparent affinity of the group 1 mutants for regulatory Ca 2 1 was decreased. The responses of all mutant exchangers to Ca 2 1 application or removal were markedly accelerated. Na 1 -dependent inactivation and regulation by Ca 2 1 are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na 1 -induced inactivated state, but that the XIP region is also involved in regulation by Ca 2 1 . key words: Na 1 -Ca 2 1 exchange • exchanger inhibitory peptide • mutagenesis • giant excised patch i n t r o d u c t i o n The sarcolemmal Na 1 -Ca 2 1 exchanger is the primary Ca 2 1 extrusion mechanism in cardiac myocytes and has a central role in regulating myocardial contractility. The exchanger has been cloned (Nicoll et al., 1990) and is modeled to consist of two groups of transmembrane segments separated by a large intracellular loop. The intracellular loop is over 500 amino acids in length but is not essential for transport function. The loop, however, has been shown to be involved in the regulation of Na 1 -Ca 2 1 exchange activity (Matsuoka et al., 1993; Matsuoka et al., 1995). The exchanger is subject to two forms of intrinsic regulation designated I 1 and I 2 (Hilgemann et al., 1992 a,b ). I 1 was first observed by Hilgemann (1990) and has also been termed Na 1 -dependent inactivation. Na 1 -dependent inactivation is manifested as a partial inactivation of outward exchange current upon application of Na 1 to the intracellular surface. I 2 , also termed Ca 2 1 -dependent regulation, was first described in the squid axon (DiPolo, 1979). The exchanger is regulated by intracellular Ca 2 1 at a high affinity binding site which is distinct from the Ca 2 1 transport site. Regulatory Ca 2 1 is not transported. Binding of regulatory Ca 2 1 activates Na 1 -Ca 2 1 exchange activity with an apparent K D of 0.2–0.4 m M (Matsuoka et al., 1995) although this measurement is dependent on experimental conditions. The binding site for regulatory Ca 2 1 has been identified on a central region of the large intracellular loop (Levitsky et al., 1994; Matsuoka et al., 1995). Regulation by both Na 1 and Ca 2 1 are most readily studied using the giant excised patch technique (Hilgemann, 1989). Thus, for example, with a membrane patch in the inside-out configuration and with Ca 2 1 in the pipette, rapid addition of Na 1 to the bath initiates an outward Na 1 -Ca 2 1 exchange current; the current rapidly peaks and then decays, due to Na 1 -dependent inactivation, to a steady-state level. The decay occurs over several seconds. The inactivated state of the exchanger is modeled to form after Na 1 binds to the intracellular transport site of the protein. Three Na 1 ions bind to the transport site and either induce translocaAddress correspondence to D. Nicoll, Cardiovascular Research Laboratory, MRL 3645, 675 Circle Drive South, Los Angeles, CA 900951760. Fax: 310-206-5777; E-mail: [email protected] on M ay 8, 2017 D ow nladed fom Published February 1, 1997