Structure-function relationships of K+-dependent Na+/Ca2+ exchangers (NCKX).

K+-dependent Na+/Ca2+ exchanger proteins (NCKX1-5) of the SLC24 gene family play important roles in a wide range of biological processes including but not limited to rod and cone photoreceptor vision, olfaction, enamel formation and skin pigmentation. NCKX proteins are also widely expressed throughout the brain and NCKX2 and NCKX4 knockouts in mice have specific phenotypes. Here we review our work on structure-function relationships of NCKX proteins. We discuss membrane topology, domains critical to transport function, and residues critical to cation binding and transport function, all in the context of crystal structures that were obtained for the archaeal Na+/Ca2+ exchanger NCX_Mj.

Cellular localization of the K+ -dependent Na+ -Ca2+ exchanger NCKX5 and the role of the cytoplasmic loop in its distribution in pigmented cells.

NCKX5 is a bidirectional K+ -dependent Na+ -Ca2+ exchanger, which belongs to the SLC24A gene family. In particular, the A111T mutation of NCKX5 has been associated with reduced pigmentation in European populations. In contrast to other NCKX isoforms, which function in the plasma membrane (PM), NCKX5 has been shown to localize either in the trans-Golgi network (TGN) or in melanosomes. Moreover, sequences responsible for retaining its intracellular localization are unknown. This study addresses two major questions: (i) clarification of intracellular location of NCKX5 and (ii) identification of sequences that retain NCKX5 inside the cell. We designed a set of cDNA constructs representing NCKX5 loop deletion mutants and NCKX2-NCKX5 chimeras to address these two questions after expression in pigmented MNT1 cells. Our results show that NCKX5 is not a PM resident and is exclusively located in the TGN. Moreover, the large cytoplasmic loop is the determinant for retaining NCKX5 in the TGN.

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

K(+)-dependent Na(+)/Ca(2+) exchangers belong to the solute carrier 24 (SLC24A1-5) gene family of membrane transporters. Five different gene products (NCKX1-5) have been identified in humans, which play key roles in biological processes including vision, olfaction, and skin pigmentation. NCKXs are bi-directional membrane transporters that transport 1 Ca(2+)+K(+) ions in exchange for 4 Na(+) ions. Recent studies have linked mutations in the SLC24A4 (NCKX4) and SLC24A5 (NCKX5) genes to amylogenesis imperfecta (AI) and non-syndromic oculocutaneous albinism (OCA6), respectively. Here, we introduced mutations found in patients with AI and OCA6 into human SLC24A4 (NCKX4) cDNA leading to single residue substitutions in the mutant NCKX4 proteins. We measured NCKX-mediated Ca(2+) transport activity of WT and mutant NCKX4 proteins expressed in HEK293 cells. Three mutant NCKX4 cDNAs represent mutations found in the SCL24A4 gene and three represent mutations found in the SCL24A5 gene involving residues conserved between NCKX4 and NCKX5. Five mutant proteins had no observable NCKX activity, whereas one mutation resulted in a 78% reduction in transport activity. Total protein expression and trafficking to the plasma membrane (the latter with one exception) were not affected in the HEK293 cell expression system. We also analyzed two mutations in a Drosophila NCKX gene that have been reported to result in an increased susceptibility for seizures, and found that both resulted in mutant proteins with significantly reduced but observable NCKX activity. The data presented here support the genetic analyses that mutations in SLC24A4 and SLC24A5 are responsible for the phenotypic defects observed in human patients.

The Role of Phospholipase D in Regulated Exocytosis.

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.

Cholesterol-independent effects of methyl-ß-cyclodextrin on chemical synapses.

The cholesterol chelating agent, methyl-ß-cyclodextrin (MßCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MßCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MßCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MßCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MßCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MßCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MßCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MßCD can affect both presynaptic and postsynaptic properties, and that some effects of MßCD are unrelated to cholesterol chelation.

Anionic lipids in Ca(2+)-triggered fusion.

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca(2+)-sensing and even bind Ca(2+) directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca(2+) sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion.

Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca(2+)-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca(2+)-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

Identifying critical components of native Ca2+-triggered membrane fusion. Integrating studies of proteins and lipids.

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca(2+)-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca(2+) sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca(2+) sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca(2+) sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca(2+)-triggered membrane fusion.