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M Shigekawa

Publications and source records attributed to M Shigekawa.

At least 37 records · Page 2Linked to original sources
PubMed2000

Physiological functions of the regulatory domains of the cardiac Na(+)/Ca(2+) exchanger NCX1.

Physiological functions of the intracellular regulatory domains of the Na(+)/Ca(2+) exchanger NCX1 were studied by examining Ca(2+) handling in CCL39 cells expressing a low-affinity Ca(2+) regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na(+) inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Delta246-672). We found that D447V/D498I was unable to efficiently extrude Ca(2+) from the cytoplasm, particularly during a small rise in intracellular Ca(2+) concentration induced by the physiological agonist alpha-thrombin or thapsigargin. The same mutant took up Ca(2+) much less efficiently than the wild-type NCX1 in Na(+)-free medium when transfectants were not loaded with Na(+), although it appeared to take up Ca(2+) normally in transfectants preloaded with Na(+). XIP-4YW and, to a lesser extent, Delta246-672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na(+)-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca(2+) handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.

Adenosine Triphosphate↗
PubMed1999

Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+).

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.

Amino Acid Sequence↗
PubMed1999

Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal alpha-1 and alpha-2 repeats. Unexpectedly, we found that the connecting loop in the alpha-1 repeat forms a re-entrant membrane loop with both ends facing the extracellular side and one residue (Asn-125) being accessible from the inside and that the region containing the alpha-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Animals↗
PubMed1998

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation.

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.

Amino Acid Sequence↗
PubMed1998

Alpha1-syntrophin has distinct binding sites for actin and calmodulin.

Overlay and co-sedimentation assays using recombinant alpha1-syntrophin proteins revealed that two regions of alpha1-syntrophin, i.e. aa 274-315 and 449-505, contain high-affinity binding sites for F-actin (Kd 0.16-0.45 microM), although only a single high-affinity site (Kd 0.35 microM) was detected in the recombinant full-length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti-syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin-glycoprotein complex or for other cell functions. We also found that alpha1-syntrophin binds calmodulin at two distinct sites with high (Kd 15 nM) and low (Kd 0.3 microM) affinity.

Actins↗
PubMed1998

Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes.

The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-alpha-sarcoglycan co-precipitated integrin alpha 5 beta 1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, alpha-sarcoglycan, integrin alpha 5 beta 1, and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of alpha- and gamma-sarcoglycans but not beta-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with alpha- and gamma-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of alpha- and gamma-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.

Animals↗
PubMed1998

Na+/Ca2+ exchanger overexpression impairs calcium signaling in fibroblasts: inhibition of the [Ca2+] increase at the cell periphery and retardation of cell adhesion.

We examined the Ca2+ handling property and cell function of CCL39 fibroblasts highly overexpressing the cardiac isoform (NCX1) of Na+/ Ca2+ exchanger. In NCX1 transfectants in 146 mM Na+, ionomycin, alpha-thrombin or thapsigargin only produced a small transient increase in [Ca2+]i compared to the large increase seen in control cells, although resting [Ca2+]i was not significantly different between these cells. In Na+-free medium, in contrast, the [Ca2+]i responses in NCX1 transfectants and control cells stimulated with these agents were not different, indicating that the Ca2+ content of the intracellular store(s) does not decrease on NCX1 transfection. The expression levels of the endoplasmic reticulum and plasma membrane Ca2+-ATPases, and thrombin- or serum-stimulated cell growth were not altered in NCX1 transfectants. The latter finding suggests that Ca2+ signaling in the nucleus is not impaired appreciably. On fluorescence imaging and confocal microscopy, we found that [Ca2+] did not increase in the peripheral cytoplasm of these cells treated with alpha-thrombin in Na+-containing medium. In these NCX1 transfectants, activation of the plasma membrane Ca2+-activated K+ channels by thrombin or ionomycin was markedly suppressed, and the integrin-mediated adhesion to substrate was significantly delayed compared with control cells. NCX1-overexpressing CCL39 cells thus seem to be a good model with which we can study the Ca2+-regulated membrane processes under physiologically relevant conditions.

Animals↗
PubMed1998

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger.

To assess the role of Ca2+ in regulation of the Na+/H+ exchanger (NHE1),we used CCL-39 fibroblasts overexpressing the Na+/Ca2+ exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by alpha-thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with the calmodulin (CaM)-binding domain deleted (amino acids 637-656), suggesting that the effect of NCX1 transfection involves Ca2+-CaM binding. Expression of NCX1 only slightly inhibited platelet-derived growth factor BB-induced alkalinization and did not affect hyperosmolarity- or phorbol 12-myristate 13-acetate-induced alkalinization. Downregulation of protein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely in NCX1-transfected cells, suggesting that the thrombin effect is mediated exclusively via Ca2+ and PKC. On the other hand, deletion mutant study revealed that PKC-dependent regulation occurs through a small cytoplasmic segment (amino aids 566-595). These data suggest that a mechanism involving direct Ca2+-CaM binding lasts for a relatively long period after agonist stimulation, despite apparent short-lived Ca2+ mobilization, and further support our previous conclusion that Ca2+- and PKC-dependent mechanisms are mediated through distinct segments of the NHE1 cytoplasmic domain.

Animals↗
PubMed1998

Differential inhibition of Na+/Ca2+ exchanger isoforms by divalent cations and isothiourea derivative.

We compared the properties of three mammalian Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni2+ and other cations as well as the recently identified inhibitor isothiourea derivatives on intracellular Na+-dependent 45Ca2+ uptake into CCL-39 (Dede) fibroblasts stably expressing each isoform. All these NCX isoforms had similar affinities for the extracellular transport substrates Ca2+ and Na+. Ni2+ inhibited 45Ca2+ uptake by competing with Ca2+ for the external transport site, with 10-fold less affinity in NCX3 than in NCX1 or NCX2. Ni2+ and Co2+ were most efficient in such discrimination of NCX isoforms, although their inhibitory potencies were less than those of La3+ and Cd2+. The monovalent cation Li+ stimulated 45Ca2+ uptake rate by all NCX isoforms similarly with low affinity, although the extent of stimulation was somewhat smaller in NCX1. On the other hand, the isothiourea derivative KB-R7943 was threefold more inhibitory to NCX3 than to NCX1 or NCX2. Thus distinct differences in the kinetic and pharmacological properties were detected between NCX3 and the other two isoforms.

Animals↗
PubMed1997

Calmodulin-binding autoinhibitory domain controls "pH-sensing" in the Na+/H+ exchanger NHE1 through sequence-specific interaction.

The calmodulin (CaM)-binding domain reduces the affinity of the Na+/H+ exchanger NHE1 for intracellular H+ by exerting an autoinhibitory function in quiescent cells. We replaced this domain (aa 637-656) with homologous segments from other NHE isoforms (NHE2 and 4) or functionally similar regions from other sources (Na+/Ca2+ exchanger, CaM-dependent protein kinase II, plasma membrane Ca2+-pump, or CaM-binding peptide Trp3). The NHE-1-, NHE2-, and NHE4-segments bound CaM with Kds of 16, 130, and 27 nM, respectively. These chimeric molecules were expressed in the exchanger-deficient cell PS120. NHE1 with incorporated NHE2-segment was activated in response to Ca2+-mobilizing agents ionomycin and thrombin resulting in an alkaline shift of the intracellular pH (pHi)-dependence of 22Na+ uptake, as was the case with the intact rat NHE2. In contrast, incorporation of the NHE4-segment or other CaM-binding segments induced a constitutive alkaline shift of pHi-dependence with concomitant abolishment of Ca2+-dependent activation, indicating that these segments could not function as an autoinhibitory domain in NHE1. Detailed analyses revealed that Leu639, Lys651 and Tyr652, conserved in the NHE1- and NHE2-segments, but not in the NHE4-segment, are important for the autoinhibition. Furthermore, 125I-labeled CaM-binding peptide from NHE1 was efficiently crosslinked to the NHE1 protein, suggesting that the inhibitory domain physically interacts with part(s) of the molecule. Together, these findings support the notion that the reduction of H+ affinity in Na+/H+ exchange occurs through a mechanism involving a highly sequence-specific interaction of the inhibitory domain with its putative acceptor in NHE1.

Amino Acid Sequence↗
PubMed1997

Whole-cell currents from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell CCL39.

A conventional patch-clamp technique was used to record the whole-cell current from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell. Ca2+ was extracellularly applied to the Na+-loaded cell to activate the outward current by operating the reverse mode of NCX1. No measurable outward current was ever elicited from the nontransfected cell. Na+/Ca2+ exchange blocker 5 mM Ni2+ or 3 microM KB-R7943 that was applied extracellularly abolished the outward current. With 140 mM external Li+ (replacing Na+), the outward current was transient during the Ca2+ application. In contrast, with 140 mM external Na+, the outward current was maintained without any inactivation during the Ca2+ application. I-V relations predicted from the whole-cell clamp protocols used were obtained both before and during the Ca2+ application. The exchanger whole-cell currents are thus successfully detectable from NCX1 which is overexpressed in this stable transfectant system.

Animals↗
PubMed1997

Identification of cytoplasmic subdomains that control pH-sensing of the Na+/H+ exchanger (NHE1): pH-maintenance, ATP-sensitive, and flexible loop domains.

To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminated cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pHi-sensitivity of these mutants by measuring the resting pHi in cells placed in an acidic medium (pH 6.0) and pHi-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pHi-sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pHi-sensitivity. Subdomain I had a pHi-maintenance function, preserving pHi-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pHi-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile "flexible loop," permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the "H(+)-modifier site.".

Adenosine Triphosphate↗
PubMed1997

Molecular physiology of vertebrate Na+/H+ exchangers.

This review describes recent progress concerning the molecular aspects of the Na+/H+ exchanger. The Na+/H+ exchanger is an important regulator for intracellular pH, cell volume, and transepithelial Na+ transport. It exists in virtually all cells with cell type-dependent pattern of isoform expression, and it is regulated in response to a variety of extracellular stimuli, among them not only agonists such as growth factors and hormones but also mechanical stimuli such as osmotic stress and cell spreading. Thus this transporter is also an excellent model to study the signal transduction. Since the first molecular cloning of the Na+/H+ exchanger, detailed studies revealed many interesting features of this transporter. At present, at least five different isoforms of the Na+/H+ exchanger are known. These isoforms differ in tissue localization, sensitivity of inhibitors, and mode of transcriptional and posttranscriptional regulation, allowing them to participate in different physiological processes. We have only started to understand an intriguing mechanism underlying these functional differences among the exchanger isoforms. Because the Na+/H+ exchanger is relatively simple in terms of its kinetic features, e.g., a simple 1:1 stoichiometry of Na+ and H+ and no input of metabolic energy such as ATP hydrolysis, the study of its structural and mechanistic aspects would also serve as a good model to understand the general mechanism of various ion transporters.

Animals↗
PubMed1997

mRNA expression and cDNA sequences of beta- and gamma-sarcoglycans are normal in cardiomyopathic hamster heart.

In BIO14.6 cardiomyopathic hamster heart, the dystrophin-glycoprotein complex is disrupted and sarcoglycans are greatly reduced in abundance. We examined whether the gene expression of beta- and gamma-sarcoglycans is indeed defective in this hamster. We found that mRNA expression for these proteins and the cDNA sequences of their coding regions are identical in both normal and myopathic hamster cardiomyocytes. The results strongly suggest that defect in a currently unknown sarcoglycan-associated protein(s) is responsible for the deficiency of the sarcoglycan complex that leads to muscle cell necrosis in the myopathic hamster.

Amino Acid Sequence↗
PubMed1996

Generation of cell transfectants expressing cardiac calcium ion channel and calcium indicator protein aequorin.

Chinese hamster ovary (CHO) cells stably coexpressing cardiac calcium ion channel [L-type calcium channel or ryanodine receptor (RyR)] and the calcium-sensitive bioluminescent protein aequorin were generated by transfecting aequorin cDNA. In a selected clone, C1-17, carrying the L-type calcium channel, depolarization induced by high concentration of K+ produces aequorin luminescence. In another clone, R3-7, carrying RyR, caffeine produces aequorin luminescence. In the presence of selective calcium ion channel blockers, the aequorin luminescence was inhibited in a dose-dependent manner. These results indicate that functionally expressed calcium ion channels in these transformants can be monitored through the activation of endogenous aequorin luminescence following a physiological signal similar to that of native calcium channel. Moreover, the aequorin system compared very well with Fura-2 measurements. Thus, the recombinant cell models, which expressed cloned calcium channel and aequorin, will contribute to the elucidation of Ca2+ movement through the cell surface and intracellular calcium ion channels.

Aequorin↗
PubMed1996

A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1.

No.7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a selective inhibitor of the Na+/Ca2+ exchanger (NCX1), has been newly synthesized. It dose-dependently inhibited Na+i-dependent 45Ca2+ uptake and Na+i-dependent [Ca2+]i increase in cardiomyocytes, smooth muscle cells, and NCX1-transfected fibroblasts (IC50 = 1.2-2.4 microM). Inhibition was observed without prior incubation with the agent and was completely reversed by washing cells with buffer for 1 min. Interestingly, No.7943 was much less potent in inhibiting Na+o-dependent 45Ca2+ efflux and Na+o-induced [Ca2+]i decline (IC50 = >30 microM), indicating that it selectively blocks the reverse mode of Na+/Ca2+ exchange in intact cells. In cardiac sarcolemmal preparations consisting mostly of inside-out vesicles, the agent inhibited Na+i-dependent 45Ca2+ uptake and Na+o-dependent 45Ca2+ efflux with similar, but slightly lower, potencies (IC50 = 5.4-13 microM). Inhibition was noncompetitive with respect to Ca2+ and Na+ in both cells and sarcolemmal vesicles. These results suggest that No.7943 primarily acts on external exchanger site(s) other than the transport sites in intact cells, although it is able to inhibit the exchanger from both sides of the plasma membrane. No.7943 at up to 10 microM does not affect many other ion transporters nor several cardiac action potential parameters. This agent at these concentrations also did not influence either diastolic [Ca2+]i or spontaneous beating in cardiomyocytes. Furthermore, No.7943 markedly inhibited Ca2+ overloading into cardiomyocytes under the Ca2+ paradox conditions. Thus, No.7943 is not only useful as a tool with which to study the transport mechanism and physiological role of the Na+/Ca2+ exchanger but also has therapeutic potential as a selective blocker of excessive Ca2+ influx mediated via the Na+/Ca2+ exchanger under pathological conditions.

Animals↗
PubMed1996

Phosphorylation-dependent regulation of cardiac Na+/Ca2+ exchanger via protein kinase C.

The cardiac Na+/Ca2+ exchanger (NCX1) plays a major role in the extrusion of Ca2+ from cardiomyocytes. We studied the role of protein phosphorylation in the regulation of cardiac NCX1 using CCL39 stably overexpressing the canine cardiac NCX1 and rat neonatal cardiomyocytes. In both cell types, the NCX1 protein immunoprecipitated with a chicken anti-NCX1 antibody exhibited a significant basal phosphorylation that was further enhanced by treatment with endothelin-1, acidic fibroblast growth factor, phorbol 12-myristate 13-acetate, or okadaic acid. In contrast, calphostin C, K252a, or EGTA inhibited the phosphorylation. The phosphorylation occurred on two major tryptic phosphopeptides (P1 and P2) exclusively on serine residues. Evidence is presented suggesting that P2 was derived from an N-terminal half (amino acids 240-475) of the central cytoplasmic domain of NCX1 and was phosphorylated directly by protein kinase C (PKC). The agents that increased NCX1 phosphorylation significantly enhanced both the forward and reverse modes of Na+/Ca2+ exchange. This exchange activation exhibited a very good correlation with the NCX1 phosphorylation. In NCX1-transfected cells, PKC down-regulation following prolonged exposure to phorbol 12-myristate 13-acetate abolished the acidic fibroblast growth factor-induced activation of exchange activity. On the other hand, cell ATP depletion reduced the exchange activity and abolished the effects of the above agents on exchange activity. These results indicate that the cardiac NCX1 is up-regulated by PKC-catalyzed phosphorylation. The cardiac NCX1 thus could play an important role in the previously reported negative inotropic actions of phorbol esters and other PKC-activating agents.

Adenosine Triphosphate↗