Molecular Cloning of a Fourth Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family, NCKX4

Abstract

We report here the identification and characterization of a fourth member of the potassium-dependent sodium-calcium exchanger gene family, NCKX4 (gene SLC24A4), which mapped to the chromosomal region 14q32. Human NCKX4 encoded a protein of 605 amino acids that displayed a high level of sequence identity to previously described family members, rod NCKX1 (gene SLC24A1), cone/neuronal NCKX2 (gene SLC24A2), and ubiquitous NCKX3 (gene SLC24A3), in the hydrophobic regions surrounding the ␣-repeat sequences thought to form the ion-binding pocket used for transport. The protein product of the NCKX4 gene shared the highest level of amino acid identity, as well as an almost identical arrangement of exon boundaries, with NCKX3, indicating that these two genes have arisen from a recent duplication event. NCKX4 transcripts were abundantly expressed in all brain regions, aorta, lung, and thymus, as well as at a lower level in many other tissues. The NCKX4 protein demonstrated potassium-dependent sodium calcium exchanger activity when assayed in transfected HEK293 cells using digital imaging of fura-2 fluorescence. The discovery of NCKX4, as far as can be ascertained from the current version of the human genome sequence, completes the mammalian potassiumdependent sodium-calcium exchanger gene family.Sodium-calcium exchange is an important determinant of intracellular Ca 2ϩ control. Detailed structural and functional studies have revealed an exchanger gene superfamily comprising two arms: the potassium-independent sodium-calcium exchangers (NCX) 1 and potassium-dependent sodium-calcium exchangers (NCKX) (1-3). The protein products of these two family branches share sequence similarity in two internally homologous, hydrophobic, domains commonly referred to as the ␣-repeats (4). NCX proteins are thought to catalyze the extrusion of one intracellular Ca 2ϩ ion in exchange for three or four extracellular Na ϩ ions (1, 5, 6). On the other hand, NCKX proteins are thought to transport one intracellular Ca 2ϩ and one K ϩ ion in exchange for four extracellular Na ϩ ions (7-10). Three NCX genes (NCX1 (SLC8A1), NCX2 (SLC8A2), and NCX3 (SLC8A3)) whose protein products share a high degree of sequence identity, especially within the transmembrane spanning domains, have been cloned (11-13). NCX1 is widely distributed in many different mammalian tissues and cell types and is driven by three tissue-specific promoters (14 -17), whereas NCX2 and NCX3 are only expressed in brain and skeletal muscle (12, 13). The functional role of the NCX family is best exemplified by the much studied mammalian cardiac NCX1, which plays a crucial role in the relaxation process of heart muscle by extruding the Ca 2ϩ that enters at the beginning of systole. The physiological role(s) of NCX1 and of the other NCX family members in tissues other than the heart has recently attracted considerable attention (1).Three genes of the NCKX family have also been cloned. NCKX1 (SLC24A1) was initially characterized in retinal rod outer segments and first cloned from bovine retin...

A Functional Study of Mutations in K+-dependent Na+-Ca2+ Exchangers Associated with Amelogenesis Imperfecta and Non-syndromic Oculocutaneous Albinism

“…NCKX4 and NCKX-X are trafficked to the plasma membrane and operate there as K ϩ -dependent Na ϩ /Ca 2ϩ exchangers (24,25). In contrast, the NCKX5 protein is not found in the plasma membrane but has been reported to be localized in the melanosomal membranes (26) or the Trans-Golgi network (27).…”

K+-Dependent Na+/Ca2+Exchangers: Key Contributors to Ca2+Signaling

“…NCKX1 is exclusively expressed in retinal rod outer segments, whereas NCKX2 is expressed in brain and cone photoreceptors (47,48,59). NCKX3 and 4 display a broader pattern of tissue expression, although they are also prominently expressed in the brain (31,36,39). These observations indicate that NCKX2, 3, and 4 are likely to play important roles in neuronal Ca 2+ signaling.…”

“…The distributions of NCKX3 and 4 in mouse brain have also been investigated by in situ hybridization and were also found to be restricted to neuronal cell bodies (31,36). NCKX3 displayed particularly strong staining throughout the thalamus, CA1 hippocampal neurons, and cortical layer IV, whereas NCKX4 expression was restricted to the anterodorsal nucleus of the thalamus, throughout the hippocampal neurons, and in cortical layers II to VI.…”

“…Based on sequence identity, the other NCKX family members are expected to display the same stoichiometry. Recombinant NCKX3 and 4 have been demonstrated to be electrogenic (31,36) and were recently shown to display similar apparent affinities for Ca 2+ and Na + as NCKX2 but much higher affinities for K + (~1 mM) (61).…”

“…The regions encoding the large cytosolic loop of NCX1 are alternatively spliced to yield protein products that are subject to differential functional regulation by cations (13,14). NCKX2 and 4 were found to be alternatively spliced, with the longer forms possessing an additional 17 or 19 amino acid residues, respectively, in the large cytosolic loops, although there are no obvious functional consequences and no detailed analyses of possible differential regulation have been performed (36,47,59). However, NCKX2 was recently shown to become inactivated in a time-and Na + -dependent manner, a phenomenon that has already been well described for NCX1 (16), suggesting that NCKX2 is also subject to ionic regulation (1).…”