The topology of the C-terminal sections of the NCX1 Na (+) /Ca ( 2+) exchanger and the NCKX2 Na (+) /Ca ( 2+) -K (+) exchanger.

Mammalian Na (+) /Ca ( 2+) (NCX) and Na (+) /Ca ( 2+) -K (+) exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na (+) /Ca ( 2+) exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.

Topologic investigation of the NCKX2 Na+/Ca2+-K+ exchanger alpha-repeats.

Algorithms suggest that NCKX proteins consist of an N-terminal signal peptide and 11 transmembrane segments divided in two groups of 5 and 6, respectively, separated by a large cytoplasmic loop. This predicted topology places the NCKX alpha-repeats with the same orientation in the plasma membrane. Using thiol-specific drug treatment and site-directed disulfide mapping, we have investigated the orientation of the NCKX2 alpha-repeats. Our results suggest that the NCKX2 alpha-repeats have an antiparallel orientation in the plasma membrane. In addition, these experiments suggest that the alpha-repeats are found in close proximity in the mature configuration of the protein.

Site-directed disulfide mapping of residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger.

The Na+/Ca2+-K+ exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na+ and K+ gradients to extrude cytoplasmic Ca2+. NCKX proteins are made up of two clusters of hydrophobic segments, both thought to consist of five putative membrane-spanning alpha-helices, and separated by a large cytoplasmic loop. The two most conserved regions within the NCKX sequence are known as the alpha1 and alpha2 repeats, and are found within the first and second set of transmembrane domains, respectively. The alpha repeats have previously been shown to contain residues critical for transport function. Here we used site-directed disulfide mapping to report that the alpha repeats are found in close proximity in three-dimensional space, bringing together key functional NCKX residues, e.g., the two critical acidic residues, Glu188 and Asp548. Glu188Cys in the alpha1 repeat could form a disulfide cross-link with Asp548Cys in the alpha2 repeat. Surprisingly, cysteine substitutions of Ser185 in the alpha1 repeat could form disulfide cross-links with cysteine substitutions of three residues in the alpha2 repeat (Ser545, Asp548, and Ser552), thought to cover close to two full turns of an alpha helix, implying an area of increased flexibility. Using the same method, Asp575, a residue critical for the K+ dependence of NCKX, was shown to be in the proximity of Ser185 and Glu188, consistent with its role in enabling K+ to bind to a single Ca2+ and K+ binding pocket.

Residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger.

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) from cells utilizing both the inward Na(+) gradient and the outward K(+) gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).

Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger.

Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.

Assembly of retinal rod or cone Na(+)/Ca(2+)-K(+) exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs.

Proper control of intracellular free Ca(2+) is thought to involve subsets of proteins that co-localize to mediate coordinated Ca(2+) entry and Ca(2+) extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca(2+) influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na(+)/Ca(2+)-K(+) exchanger (NCKX) is the only Ca(2+) extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments.

TUC-4b, a novel TUC family variant, regulates neurite outgrowth and associates with vesicles in the growth cone.

The TUC (TOAD-64/Ulip/CRMP) proteins are homologs of UNC-33, a protein that is required for axon extension and guidance in Caenorhabditis elegans. The TUC proteins are expressed in newly born neurons in the developing nervous system and have been implicated in semaphorin signaling and neuronal polarity. Here, we identify several new variants of the TUC family, each of which is expressed during distinct periods of neural development. We cloned and characterized TUC-4b, a variant of TUC-4a that includes a unique N-terminal extension. The functional relevance of this N-terminal domain is demonstrated by the finding that overexpression of TUC-4b, but not TUC-4a, results in increased neurite length and branching. Furthermore, whereas TUC-4a is expressed throughout life, TUC-4b is expressed exclusively during embryonic development. TUC-4b is localized to SV2 (synaptic vesicle protein 2)-positive vesicles in the central domain of the growth cone, suggesting a potential role in growth cone vesicle transport. Furthermore, TUC-4b interacts with the SH3A (Src homology 3A) domain of intersectin, an endocytic-exocytic adaptor protein. Together, these data suggest that TUC-4b can regulate neurite extension and branching through a mechanism that may involve membrane transport in the growth cone.

Topology of the retinal cone NCKX2 Na/Ca-K exchanger.

The Na/Ca-K exchanger (NCKX) is a polytopic membrane protein that plays a critical role in Ca(2+) homeostasis in retinal rod and cone photoreceptors. The NCKX1 isoform is found in rods, while the NCKX2 isoform is found in cones, in retinal ganglion cells, and in various parts of the brain. The topology of the Na/Ca-K exchanger is thought to consist of two large hydrophilic loops and two sets of transmembrane spanning segments (TMs). The first large hydrophilic loop is located extracellularly at the N-terminus; the other is cytoplasmic and separates the two sets of TMs. The TMs consist of either five and five membrane spanning helices or five and six membrane spanning helices, depending upon the predictive algorithm used. Little specific information is yet available on the orientation of the various membrane spanning helices and the localization of the short loops connecting these helices. In this study, we have determined which of the connecting loops are exposed to the extracellular milieu using two different methods: accessibility of substituted cysteine residues and insertion of N-glycosylation sites. The two methods resulted in a consistent NCKX topology in which the two sets of TMs each contain five membrane spanning helices. Our new model places what was previously membrane spanning helix six in the cytoplasm, which places the C-terminus on the extracellular surface. Surprisingly, this NCKX topology model is different from the current NCX topology model with respect to the C-terminal three membrane helices.

Scanning mutagenesis of the alpha repeats and of the transmembrane acidic residues of the human retinal cone Na/Ca-K exchanger.

The Na/Ca-K exchanger (NCKX) utilizes the inward sodium gradient and outward potassium gradient for Ca(2+) extrusion; two distinct NCKX isoforms are expressed in the outer segments of retinal rod (NCKX1) and cone (NCKX2) photoreceptors, respectively, where NCKX extrudes Ca(2+) that enters photoreceptors via the cGMP-gated channels. We carried out the first systematic NCKX mutagenesis study in which 96 residues were mutated in the human cone NCKX2 cDNA, and functional consequences of these mutations were measured; the residues selected for mutagenesis are conserved between rod and cone NCKX, the large majority are also conserved in NCKX paralogs found in lower organisms, and finally, they include the few residues conserved between members of the NCKX and members of the NCX (potassium-independent Na/Ca exchange) gene families. Twenty-five residues were identified for which mutagenesis reduced NCKX function to <20% of wild-type cone NCKX2 activity, while protein expression and plasma membrane targeting were not affected. Three classes of residues were found to be most sensitive toward mutagenesis: acidic (glutamate/aspartate) residues, polar (serines/threonine) residues, and glycine residues. These results are discussed with respect to residues that may contribute to the NCKX cation binding site(s).

The Na/Ca-K exchanger gene family.

Ca(2+) extrusion driven by both the inward Na(+) gradient as well as the outward K(+) gradient is essential for visual transduction in retinal rod and cone photoreceptors because it removes Ca(2+) that enters photoreceptors via the cGMP-gated and light-sensitive channels. We have cloned rod and cone Na/Ca-K exchanger (NCKX) cDNAs from several species, and we have cloned NCKX cDNAs from lower organisms that lack vertebrate-type vision. Although in situ NCKX physiology has only been documented for vertebrate photoreceptors, it is now clear that NCKX gene products have a much broader distribution pattern. Here, we review some of the structural and functional features that have emerged from our studies on different members of the NCKX gene family.