We describe here the identification and characterization of a novel member of the family of K ؉ -dependent Na ؉ /Ca 2؉ exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "␣ -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K ؉ -dependent Na ؉ /Ca 2؉ exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive 45 Ca 2؉ uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K ؉ -dependent Na ؉ /Ca 2؉ exchanger family and suggests this class of transporter has a more widespread role in cellular Ca 2؉ handling than previously appreciated.

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...

Ryr1 I4895T/wt (IT/؉) mice express a knockin mutation corresponding to the human I4898T EC-uncoupling mutation in the type 1 ryanodine receptor/Ca 2؉ release channel (RyR1), which causes a severe form of central core disease (CCD). IT/؉ mice exhibit a slowly progressive congenital myopathy, with neonatal respiratory stress, skeletal muscle weakness, impaired mobility, dorsal kyphosis, and hind limb paralysis. Lesions observed in myofibers from diseased mice undergo age-dependent transformation from minicores to cores and nemaline rods. Early ultrastructural abnormalities include sarcomeric misalignment, Z-line streaming, focal loss of cross-striations, and myofibrillar splitting and intermingling that may arise from defective myofibrillogenesis. However, manifestation of the disease phenotype is highly variable on a Sv129 genomic background. Quantitative RT-PCR shows an equimolar ratio of WT and mutant Ryr1 transcripts within IT/؉ myofibers and total RyR1 protein expression levels are normal. We propose a unifying theory in which the cause of core formation lies in functional heterogeneity among RyR1 tetramers. Random combinations of normal and either leaky or EC-uncoupled RyR subunits would lead to spatial differences in Ca 2؉ transients; the resulting heterogeneity of contraction among myofibrils would lead to focal, irreversible tearing and shearing, which would, over time, enlarge to form minicores, cores, and nemaline rods. The IT/؉ mouse line is proposed to be a valid model of RyR1-related congenital myopathy, offering high potential for elucidation of the pathogenesis of skeletal muscle disorders arising from impaired EC coupling.calcium ͉ central core disease ͉ multiminicore disease ͉ nemaline rod myopathy ͉ ryanodine receptor

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca 2+ release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1 I4895T mutation was introduced by using a “knockin” protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca 2+ release is absent, although retrograde enhancement of DHPR Ca 2+ conductance is retained. IT/IT mice, in which RyR1-mediated Ca 2+ release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca 2+ signaling in mammalian embryogenesis.