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K D Philipson

Publications and source records attributed to K D Philipson.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger.

We have examined the role of conserved regions and acidic or basic residues located in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger by site-directed mutagenesis. The alpha-1 and alpha-2 repeats are transmembrane regions of internal similarity, which are highly conserved among Na+-Ca2+ exchangers. We find that Na+-Ca2+ exchange activity is highly sensitive to mutagenesis in the alpha-repeats. Mutation at residues Ser-109, Ser-110, Glu-113, Ser-139, Asn-143, Thr-810, Ser-811, Asp-814, Ser-818, or Ser-838 resulted in loss of exchanger activity. Mutation at residues Thr-103, Gly-108, Pro-112, Glu-120, Gly-138, Gly-809, Gly-837, and Asn-842 resulted in reduced exchanger activity, and altered current-voltage relationships were observed with mutations at residues Gly-138 and Gly-837. Only mutation at residue Ser-117 appeared to leave exchanger activity unaffected. Thus, the alpha-repeats appear to be important components for ion binding and translocation. Another region implicated in exchanger function is a region of similarity to the Na+,K+ pump (Nicoll, D. A., Longoni, S., Philipson, K. D. (1990) Science 250, 562-565). Mutations at two residues in the pump-like region, Glu-199 and Thr-203, resulted in nonfunctional exchangers, while mutation at two other residues, Glu-196 and Gly-200, had no effect. The role of acidic and basic residues in the transmembrane segments was also examined. Mutation of several basic residues (Arg-42, His-744, Lys-751, Lys-797, and His-858) did not affect exchange activity. Of the acidic residues located outside of the alpha-repeat and pump-like regions (Asp-740, Asp-785, and Asp-798), only mutation at Asp-785 resulted in reduction of exchanger activity.

Amino Acid Sequence↗
PubMed1996

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+.

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1-10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.

Animals↗
PubMed1995

The cleaved N-terminal signal sequence of the cardiac Na(+)-Ca2+ exchanger is not required for functional membrane integration.

The cardiac Na(+)-Ca2+ exchanger differs from most other polytopic membrane proteins in that the amino terminus is cleaved during integration into the endoplasmic reticulum membrane. In this study, the cleaved N-terminal signal sequence of the exchanger was deleted (DelSS) or rendered uncleavable by mutation of the cleavage site (MutSS). Functional analysis of the mutants expressed in Xenopus laevis oocytes and sf9 insect cells demonstrates that DelSS exchanger catalyzes Na(+)-dependent Ca2+ transport at wild-type levels, while activity of MutSS exchanger is reduced to approximately 60% of wild-type in oocytes and 20% in sf9 cells. These results indicate that neither the presence nor the cleavage of the signal peptide is required for functional assembly of the exchanger protein in the membrane. Furthermore, these observations support the concept that internal topogenic signals play the major role in membrane insertion of the Na(+)-Ca2+ exchanger.

Animals↗
PubMed1995

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain.

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.

Amino Acid Sequence↗
PubMed1995

Distribution of the Na+/Ca2+ exchange protein in developing rabbit myocytes.

The Na+/Ca2+ exchanger is a major pathway for transmembrane flux of Ca2+ in cardiac cells. Immunolabeling in adult rabbit myocytes showed localization of the Na+/Ca2+ exchanger to the peripheral sarcolemma and especially in the T tubules. Previous studies have also demonstrated higher Na+/Ca2+ exchanger activity in fetal and newborn rabbit hearts in which the T tubular system is not completely developed. Indirect immunofluorescent studies were performed to localize the Na+/Ca2+ exchanger in isolated myocytes from immature (5, 11, 17, and 30 days) and adult rabbits. Cells were incubated with a monoclonal antibody to the exchanger followed by fluorescein-labeled goat anti-mouse antibody. It is found that at 5 days of age the immunofluorescent labeling was very intense and confined to the peripheral sarcolemma. After 11 days of age, localization of labeling followed the development of the T tubules. The exchanger appeared in the T tubules as soon as they were formed. The Na+/Ca2+ exchange protein is abundantly localized to the peripheral sarcolemma before and during the development of T tubule system.

Aging↗
PubMed1994

Molecular cloning and functional expression of the guinea pig cardiac Na(+)-Ca2+ exchanger.

The cDNA of the guinea pig cardiac Na(+)-Ca2+ exchanger was cloned from a lambda ZAP cDNA library. The deduced sequence of the protein corresponds to 970 amino acids and is 98% identical to the canine cardiac exchanger. The leader peptide region shows substantial variation among species. The cloned cDNA can induce Na(+)-Ca2+ exchange activity when in vitro transcribed cRNA is injected into Xenopus laevis oocytes.

Amino Acid Sequence↗
PubMed1994

Identification of the high affinity Ca(2+)-binding domain of the cardiac Na(+)-Ca2+ exchanger.

The cardiac sarcolemmal Na(+)-Ca2+ exchanger transports Na+ and Ca2+ but is also regulated by Ca2+ at a high affinity binding site. A large intracellular, hydrophilic loop of the exchanger has been suggested to contain the Ca(2+)-binding regulatory domain (Matsuoka S., Nicoll, D. A., Reilly, R. F., Hilgemann, D. W., and Philipson, K. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3870-3874). To localize the Ca(2+)-binding site(s), we expressed portions of the exchanger loop as fusion proteins and measured binding by 45Ca2+ overlay. The high affinity binding domain is located near the center of the loop and binds Ca2+ in a cooperative manner. K0.5 ranges from 0.3 to 3 microM in the presence of 0.2-5 mM Mg2+. The binding region (amino acids 371-508) has two highly acidic sequences, each characterized by 3 consecutive aspartic acid residues. Ca2+ affinity markedly decreases when these aspartates are mutated. The Ca(2+)-binding region does not contain an EF-hand motif. The mobilities during SDS-polyacrylamide gel electrophoresis of fusion proteins with high Ca2+ affinity differ depending on the presence or absence of Ca2+ in the gel loading buffer. The high affinity Ca(2+)-binding domain is probably responsible for the secondary Ca2+ regulation of the Na(+)-Ca2+ exchanger.

Amino Acid Sequence↗
PubMed1994

Cloning of the NCX2 isoform of the plasma membrane Na(+)-Ca2+ exchanger.

The Na(+)-Ca2+ exchanger is an important regulator of cellular Ca2+ levels, and one isoform of this transporter, NCX1, has been cloned previously (Nicoll, D.A., Longoni, S., and Philipson, K.D. (1990) Science 250, 562-565). We now report the cloning of a second isoform (NCX2) of the Na(+)-Ca2+ exchanger which was present in a rat brain cDNA library. NCX2 is predicted to code for a protein of 921 amino acids. NCX1 and NCX2 are 61 and 65% identical at the nucleotide and amino acid levels, respectively, and are the products of different genes. The genes for NCX1 and NCX2 are located on human chromosomes 2 and 14, respectively. Hydropathy profiles of the two exchangers are very similar. Transcripts of NCX2 are detected in brain and skeletal muscle. NCX2 was expressed in Xenopus oocytes and Na(+)-Ca2+ exchange activity was analyzed electrophysiologically by the giant inside-out, excised patch technique. Outward currents were evoked by the application of Na+ with the exchanger operating in the reversed mode (extracellular Ca2+ exchanging for intracellular Na+). The affinity for Na+ (30 mM) and the current-voltage relationship of NCX2 are similar to those for NCX1. Like NCX1, NCX2 is secondarily regulated by intracellular Ca2+, but the affinity of NCX2 for regulatory Ca2+ (1.5 microM) upon initial application of Na+ is lower than that of NCX1 (0.3 microM). The existence of multiple Na(+)-Ca2+ exchanger isoforms may provide flexibility for regulation and expression.

Amino Acid Sequence↗
PubMed1993

Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle.

The Na+/Ca2+ exchanger, driven by a transmembrane Na+ gradient, plays a key role in regulating Ca2+ concentration in many cells. Although the exchanger influences Ca2+ concentration, its activity in smooth muscle appears to be closely coupled to Ca2+ availability from intracellular stores. This linkage might result if the exchanger were positioned close to Ca2+ storage sites within the sarcoplasmic reticulum. To test this hypothesis we have developed methods to assess the relative three-dimensional distribution of proteins involved in Na+/K+ pumping, Na+/Ca2+ exchange, Ca2+ storage within the sarcoplasmic reticulum, and attachment of contractile filaments to the membrane in smooth muscle. Here we report that the Na+/Ca2+ exchanger is largely co-distributed with the Na+/K+ pump on unique regions of the plasma membrane in register with, and close to, calsequestrin-containing regions of the sarcoplasmic reticulum in sites distinct from the sites where contractile filaments attach to the membrane. This molecular organization suggests that the plasma membrane is divided into at least two functional domains, and appear to provide a mechanism for the strong linkage seen in smooth muscle between Na+/K+ pumping and Na+/Ca2+ exchange, and between Na+/Ca2+ exchange and Ca2+ release from the sarcoplasmic reticulum.

Animals↗
PubMed1993

Biosynthesis and initial processing of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

Based on the deduced amino-acid sequence of the cardiac Na(+)-Ca2+ exchanger, there are six potential N-linked glycosylation sites and a potential cleaved signal sequence. To study the post-translational modifications of the exchanger, in vitro translation was examined in the presence and absence of canine pancreatic microsomes. Glycosylation, detected as endoglycosidase H induced shifts in molecular size, was examined for proteins having different numbers of potential N-linked glycosylation sites by using full and partial length RNA transcripts. In the presence of microsomes, the molecular mass of the full-length clone increased from 110 to 113 kDa. Endoglycosidase H treatment led to a reduction to 108 kDa, indicating that glycosylation increases the molecular mass by approx. 5 kDa and a signal sequence of approx. 2 kDa is cleaved during processing. Analysis of molecular-mass shifts obtained with partial transcripts suggested that glycosylation occurs at position N-9. This was confirmed by site-directed mutagenesis studies. A molecular mass of approx. 120 kDa was measured for Western blots of cardiac sarcolemmal membrane or oocytes expressing the wild-type exchanger. The molecular mass was reduced by approx. 10 kDa for the N9Y mutant or from exchanger obtained from a baculovirus-infected insect cell line where glycosylation does not occur. The giant excised patch technique was used to determine the functional consequences of glycosylation. Na(+)-Ca2+ exchange current was examined in patches from oocytes expressing either the wild-type or N9Y mutant. The non-glycosylated mutant exhibited the same properties as the native exchanger with respect to voltage, sodium dependence, and the effects of chymotrypsin. The results indicate that glycosylation does not affect exchanger function in Xenopus oocytes and help to define exchanger topology.

Amino Acid Sequence↗
PubMed1993

The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin.

Na+-Ca2+ exchange is the major pathway of Ca2+ efflux during excitation-contraction coupling in cardiac muscle. The Na+-Ca2+ exchanger is present in cardiac transverse tubules with an apparent high density (Frank, J.S., Mottino, G., Reid, D., Molday, R. S., and Philipson, K.D. (1992) J. Cell Biol. 117, 337-345). The mechanism for this localization is unknown but may involve interactions with the cytoskeleton. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with the cytoskeletal protein ankyrin. On immunoblots of isolated canine cardiac sarcolemma, an antibody raised against purified rabbit red blood cell-ankyrin (RBC-ankyrin) recognized a 220-kDa protein, which is the same size as RBC-ankyrin. Alkaline extraction of sarcolemma removed this protein. The Na+-Ca2+ exchange protein, purified from recombinant baculovirus-infected insect cells, bound 125I-labeled-RBC-ankyrin with a KD of 42 +/- 3 nm. 125I-RBC-ankyrin was co-precipitated by antibodies to the Na+-Ca2+ exchanger after preincubation with solubilized cardiac sarcolemma. Myocardial ankyrin could be localized to both surface and T-tubular sarcolemma by immunofluorescence techniques. These results demonstrate that the cardiac Na+-Ca2+ exchanger binds ankyrin with high affinity. This interaction may be important for localizing the Na+-Ca2+ exchanger to specific domains of the sarcolemma.

Animals↗
PubMed1993

Initial localization of regulatory regions of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

We have analyzed the regulatory properties of the wild-type cardiac Na(+)-Ca2+ exchanger expressed in Xenopus laevis oocytes using the giant excised patch technique. The exchanger is activated by cytoplasmic application of chymotrypsin and exhibits a number of properties that can be changed or abolished by chymotrypsin treatment, including cytoplasmic Na(+)-dependent inactivation, secondary regulation by free cytoplasmic Ca2+, and inhibition by exchanger inhibitory peptide. Thus, the cloned exchanger expressed in oocytes exhibits regulatory properties similar to those of the native sarcolemmal exchanger. The exchanger protein contains a large (520 amino acids) hydrophilic domain modeled to be intracellular. The role of this region in exchanger function and regulation was examined by deletion mutagenesis. Mutants with residues 240-679 and 562-685 deleted exhibited exchange activity, indicating that this extensive region is not essential for transport function. Both mutants were stimulated by chymotrypsin treatment. Neither mutant demonstrated regulation by free cytoplasmic Ca2+ (Ca2+i) or inhibition by exchanger inhibitory peptide (XIP). However, mutant delta 562-685 but not delta 240-679 displayed Na(+)-dependent inactivation. The data suggest that the binding sites for XIP and regulatory Ca2+ may reside in the region encompassed by residues 562-685. A chimera made from renal and cardiac exchangers has normal regulatory characteristics and helps to further define these sites.

Animals↗
PubMed1993

Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies.

We used a panel of monoclonal antibodies raised against the canine cardiac Na(+)-Ca2+ exchanger expressed in Sf9 insect cells to analyze the immunoreactive domains and the topological organization of this membrane protein. Antibodies, which reacted strongly on Western blots of the recombinant protein, were used to screen an expression sublibrary composed of exchanger cDNA fragments. Positive clones thus indicated the expression of antibody binding sites. Linear epitopes, 16-155 amino acids in length, could be identified for four antibodies. One antibody recognized two neighboring, but nonoverlapping, sequences. All epitopes were localized to the large hydrophilic region of the exchanger connecting the putative transmembrane segments 5 and 6. The immunodominant region of the protein is a highly charged domain in the carboxy-terminal half of the hydrophilic region. Binding studies with the 3H-labeled high-affinity antibody R3F1 establish that the immunodominant region is located on the intracellular surface of the membrane. The same antibody was used to directly determine the membrane concentration of the exchanger in different cell types. Newborn rat heart cells contain approximately 6 x 10(5) exchanger molecules per cell. Exchanger densities in different cells seem to correlate with the Na(+)-dependent Ca2+ transport activity in the corresponding membrane vesicles.

Amino Acid Sequence↗