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M J Caplan

Publications and source records attributed to M J Caplan.

At least 19 recordsLinked to original sources
PubMed2001

Ion pumps in polarized cells: sorting and regulation of the Na+, K+- and H+, K+-ATPases.

The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na(+),K(+)-ATPase and the H(+),K(+)-ATPases are closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells, and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intramolecular interactions govern trafficking, distribution, and catalytic capacities of these proteins.

Animals↗
PubMed2001

Ion pump sorting in polarized renal epithelial cells.

The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

Animals↗
PubMed2001

The NH(2)-terminus of norepinephrine transporter contains a basolateral localization signal for epithelial cells.

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT-NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH(2)-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH(2)-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH(2)-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.

Amino Acid Sequence↗
PubMed2000

The C-terminal tail of the metabotropic glutamate receptor subtype 7 is necessary but not sufficient for cell surface delivery and polarized targeting in neurons and epithelia.

Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.

Animals↗
PubMed2000

The roles of carbohydrate chains of the beta-subunit on the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha and beta-subunits. The alpha-subunit is the catalytic subunit, and the beta-subunit is a glycoprotein stabilizing the alpha/beta complex in the membrane as a functional enzyme. There are seven putative N-glycosylation sites on the beta-subunit. In this study, we examined the roles of the carbohydrate chains of the beta-subunit by expressing the alpha-subunit together with the beta-subunit in which one, several, or all of the asparagine residues in the N-glycosylation sites were replaced by glutamine. Removing any one of seven carbohydrate chains from the beta-subunit retained the H(+),K(+)-ATPase activity. The effects of a series of progressive removals of carbohydrate chains on the H(+),K(+)-ATPase activity were cumulative, and removal of all carbohydrate chains resulted in the complete loss of H(+), K(+)-ATPase activity. Removal of any single carbohydrate chain did not affect the alpha/beta assembly; however, little alpha/beta assembly was observed after removal of all the carbohydrate chains from the beta-subunit. In contrast, removal of three carbohydrate chains inhibited the surface delivery of the beta-subunit and the alpha-subunit assembled with the beta-subunit, indicating that the surface delivery mechanism is more dependent on the carbohydrate chains than the expression of the H(+),K(+)-ATPase activity and alpha/beta assembly.

Animals↗
PubMed2000

A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase.

The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H, K-ATPase alpha subunit and the highly homologous Na,K-ATPase alpha subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase alpha subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na, K-ATPase alpha subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.

Amino Acid Sequence↗
PubMed2000

Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity.

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.

Adenosine Triphosphatases↗
PubMed2000

The cell biology of ion pumps: sorting and regulation.

The physiologic function of an ion pump is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na,K-ATPase and the gastric H,K-ATPase are two closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intra-molecular interactions govern these proteins' trafficking, distribution and catalytic capacity.

Animals↗
PubMed2000

Differential localization of human nongastric H(+)-K(+)-ATPase ATP1AL1 in polarized renal epithelial cells.

The human H(+)-K(+)-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K(+)-transporting ATPases. In concert with the structurally related gastric H(+)-K(+)-ATPase, it plays a major role in K(+) reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK(1) renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na(+)-K(+)-ATPase beta-subunit or the gastric H(+)-K(+)-ATPase beta-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the alpha-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na(+)-K(+)-ATPase beta-subunit. Cotransfection of ATP1AL1 with the gastric H(+)-K(+)-ATPase beta-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by (86)Rb(+) uptake measurements. In contrast, cotransfected LLC-PK(1) cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the alpha-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

Animals↗
PubMed1999

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).

Animals↗
PubMed1999

Regulation of myocardial glucose uptake and transport during ischemia and energetic stress.

Myocardial glucose utilization increases in response to the energetic stress imposed on the heart by exercise, pressure overload, and myocardial ischemia. Recruitment of glucose transport proteins is the cellular mechanism by which the heart increases glucose transport for subsequent metabolism. Moderate regional ischemia leads to the translocation of both glucose transporters, GLUT4 and GLUT1, to the sarcolemma in vivo. Myocardial ischemia also stimulates 5'-adenosine monophosphate-activated protein kinase, which may be a fuel gauge in the heart and other tissues signaling the need to turn on energy-generating metabolic pathways. Pharmacologic stimulation of this kinase increases cardiac glucose uptake and transporter translocation, suggesting that it may play an important role in augmenting glucose entry in the setting of ischemic or energetic stress. Thus, recent work has provided insight into the cellular and molecular mechanisms responsible for glucose uptake during energetic stress, which may lead to new approaches to the treatment of patients with coronary artery disease.

Adenosine Monophosphate↗
PubMed1999

The dimensions of the triangle of Koch in children.

We measured the dimensions of Koch's triangle in children with normal intracardiac anatomy to determine the relation between the size of the triangle of Koch and patient age, weight, height, and body surface area. We found that the dimensions of Koch's triangle varies significantly and directly with patient age and body habitus in this pediatric population.

Age Factors↗
PubMed1999

Nongastric H+,K+-ATPase: cell biologic and functional properties.

Several members of the H+,K+-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H+,K+-ATPase as well as a number of nongastric H+,K+-ATPase isoforms. Physiological studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. Although much has been learned about the pattern of H+,K+-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties of renal H+,K+-ATPases both in vitro and in situ. Our analysis of ion fluxes driven by a nongastric H+,K+-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H+,K+-ATPase isoforms may make diverse contributions to renal cation transport. We find that the activities of renal H+,K+-ATPases in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H+,K+-ATPase beta-subunit. Transgenic mice expressing a version of this protein in which the signal has been disabled show constitutively active renal K resorption. The identities of the H+,K+-ATPase isoforms that are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established.

Animals↗
PubMed1998

Additive effects of hyperinsulinemia and ischemia on myocardial GLUT1 and GLUT4 translocation in vivo.

BACKGROUND: Myocardial ischemia increases glucose uptake through the translocation of GLUT1 and GLUT4 from an intracellular compartment to the sarcolemma. The present study was performed to determine whether hyperinsulinemia causes translocation of myocardial GLUT1 as well as GLUT4 in vivo and whether there are additive effects of insulin and ischemia on GLUT1 and GLUT4 translocation. METHODS ADN RESULTS: Myocardial glucose uptake and transporter distribution were assessed by arteriovenous measurements, cell fractionation, and immunofluorescence. In fasted anesthetized dogs, hyperinsulinemia increased myocardial glucose extraction 3-fold (P<0.01) and the sarcolemmal content of GLUT4 by 90% and GLUT1 by 50% (P<0.05 for both) compared with saline infusion. In subsequent experiments, glucose uptake and transporter distribution were determined in ischemic and nonischemic regions of hearts from hyperinsulinemic animals during regional myocardial ischemia. Glucose uptake was 50% greater in the ischemic region (P<0.05). This was associated with a 20% increase in sarcolemmal GLUT1 and a 60% increase in sarcolemmal GLUT4 contents in the ischemic region (P<0.05 for both). CONCLUSIONS: Insulin stimulates myocardial glucose utilization through translocation of GLUT1 as well as GLUT4. Insulin and ischemia have additive effects to increase in vivo glucose utilization and augment glucose transporter translocation. We conclude that recruitment of both GLUT1 and GLUT4 contributes to increased myocardial glucose uptake during moderate reductions in coronary blood flow under insulin-stimulated conditions.

Animals↗
PubMed1998

ATP1AL1, a member of the non-gastric H,K-ATPase family, functions as a sodium pump.

The human ATP1AL1-encoded protein (an alpha subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase beta subunit (gH,Kbeta) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86Rb+-influx mediated by the ATP1AL1-gH,Kbeta heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same cells. In this study we find that the intracellular Na+ content in cells expressing ATP1AL1 and gH,Kbeta is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 microM ouabain. Moreover, analysis of net Na+ efflux in HEK 293 expressing the ATP1AL1-gH,Kbeta heterodimer reveals the presence of Na+ extrusion activity that is not sensitive to 1 microM ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na+ efflux in control HEK 293 cells is similarly sensitive to either 1 microM or 1 mM ouabain. Finally, 86Rb+ influx through the ATP1AL1-gH,Kbeta complex is comparable to the 1 mM ouabain-sensitive Na+ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase beta subunit in HEK 293 cells mediates primarily Na+,K+ rather than H+,K+ exchange.

Biological Transport↗
PubMed1998

Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells.

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.

Amino Acid Sequence↗
PubMed1998

Identification of sorting determinants in the C-terminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT-3.

In order to perform their physiologic functions, polarized epithelial cells must target ion transport proteins to the appropriate domains of their plasma membranes. Molecular signals responsible for polarized sorting have been identified for several membrane proteins which span the bilayer once. Most ion transport proteins are polytopic, however, and little is known of the signals responsible for the targeting of this class of polypeptides. Members of the gamma-aminobutyric acid (GABA) transporter family are polytopic membrane proteins found endogenously in both epithelial cells and neurons. We have identified narrowly defined sequences which are required for the proper accumulation of two members of this transporter family in Madin-Darby canine kidney cells. The highly homologous GABA transporter isoforms, GAT-2 and GAT-3, localize to the basolateral and apical surfaces, respectively, when expressed stably in Madin-Darby canine kidney cells. We have generated deletion constructs and chimeric transporters composed of complimentary portions of GAT-2 and GAT-3. We find that information which directs their differential sorting is present in the C-terminal cytoplasmic tails of these two polypeptides. A sequence of 22 amino acids at the C terminus of GAT-2 is required for the transporter's basolateral distribution and is capable of directing GAT-3 to the basolateral surface when appended to the C terminus of this normally apical polypeptide. The deletion of 32 amino acids from the C terminus of GAT-3 causes this transporter to become mislocalized to both surfaces. Moreover, removal of the final three amino acids of GAT-3 (THF) similarly disrupts its apical sorting. The GAT-3 C-terminal sequence resembles motifs which interact with PDZ domains, raising the possibility that the steady state distribution of GAT-3 at the apical plasmalemmal surface requires a protein-protein interaction mediated by its extreme C-terminal cytoplasmic tail. These data provide the first characterization of a protein-based signal required for the apical distribution of a membrane protein.

Amino Acid Sequence↗