Biomedical subjects

M L Jennings

Publications and source records attributed to M L Jennings.

At least 19 recordsLinked to original sources

PubMed2001

Direct estimate of 1:1 stoichiometry of K(+)-Cl(-) cotransport in rabbit erythrocytes.

This work was undertaken to obtain a direct measure of the stoichiometry of Na(+)-independent K(+)-Cl(-) cotransport (KCC), with rabbit red blood cells as a model system. To determine whether (86)Rb(+) can be used quantitatively as a tracer for KCC, (86)Rb(+) and K(+) effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated K(+) efflux into isosmotic NaCl was smaller than that for (86)Rb(+) by a factor of 0.68 +/- 0.11 (SD, n = 5). This correction factor was used in all other experiments to calculate the K(+) efflux from the measured (86)Rb(+) efflux. To minimize interference from the anion exchanger, extracellular Cl(-) was replaced with SO, and 4,4'-diisothiocyanothiocyanatodihydrostilbene-2,2'-disulfonic acid was present in the flux media. The membrane potential was clamped near 0 mV with the protonophore 2,4-dinitrophenol. The Cl(-) efflux at 25 degrees C under these conditions is approximately 100,000-fold smaller than the uninhibited Cl(-)/Cl(-) exchange flux and is stimulated approximately 2-fold by NEM. The NEM-stimulated (36)Cl(-) flux is inhibited by okadaic acid and calyculin A, as expected for KCC. The ratio of the NEM-stimulated K(+) to Cl(-) efflux is 1.12 +/- 0.26 (SD, n = 5). We conclude that K(+)-Cl(-) cotransport in rabbit red blood cells has a stoichiometry of 1:1.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Volume-sensitive K(+)/Cl(-) cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events.

The kinetics of activation and inactivation of K(+)/Cl(-) cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg(2+)) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg(2+), acid pH, and NEM.

Algorithms↗

PubMed1998

Transitional stages in the development of the rabbit renal collecting duct.

The collecting duct (CD) epithelium of the mammalian kidney is an extraordinary structure with respect to its functional changes during development and its heterogeneous composition when matured. All of the different nephron epithelia of the mammalian kidney consist of one single cell type. In contrast, the differentiated CD is composed of at least three distinct cell types [principal, alpha intercalated-, and beta intercalated cells] that are responsible for the multiple physiological functions of this kidney compartment. During development the function of the CD changes: initially, the CD ampulla serves as an embryonic inducer, while the matured epithelium plays a key role in maintaining the homeostasis of body fluids. At present the process of CD maturation is not well understood. Neither the time course of development nor the morphogenic factors leading to the heterogeneously composed epithelium are known. In the present study the differentiation of the CD epithelium was investigated using newly developed monoclonal antibodies and well-characterized antisera. The morphological changes induced during differentiation were monitored by immunohistochemistry and scanning electron microscopy. The experiments were performed on neonatal and adult rabbit kidneys. Results obtained by light microscopical techniques and scanning electron microscopy revealed that the ampullary tip can be distinguished from the ampullary neck, as well as from the maturing CD. A number of proteins that were not detectable in the ampulla were detected in the neonatal CD and were found at even higher concentrations in the adult CD (PCD8, chloride/bicarbonate exchanger). Other proteins (PCD9) were downregulated during differentiation. For the first time the transient character of the differentiation stage of the neonatal CD could be demonstrated unequivocally. Furthermore, considerable heterogeneity in protein expression patterns (PCD6 and PCD9) was demonstrated within the beta IC cell population of the mature CD.

Aging↗

PubMed1998

Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS.

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Culture of embryonic renal collecting duct epithelia in a gradient container.

During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.

Aldosterone↗

PubMed1996

Cloning of the rabbit homologue of mouse 'basigin' and rat 'OX-47': kidney cell type-specific expression, and regulation in collecting duct cells.

Monoclonal antibody '4D4' was generated against a gel-purified 43-50 kDa fraction of rabbit erythrocyte (RBC) ghosts. Immunoblots of rabbit RBCs, skeletal muscle, and kidney, and of a rabbit cortical collecting duct cell line (RC.SV3) yielded broad bands of 30-70 kDa that migrated at approximately 31 kDa after deglycosylation. In kidney sections, 4D4 labeled the basal plasma membranes of the proximal tubule, medullary thick ascending limb of Henle, cortical, medullary, and papillary collecting ducts, and papillary surface epithelium, as well as the lateral membranes of alpha and beta-type intercalated cells. Antibody 4D4 was used to clone a full-length kidney cDNA, which predicted a 31 kDa immunoglobulin-like glycoprotein with high homology to mouse 'gp42' or 'basigin', human 'M6' or 'EMMPRIN', rat 'OX-47' or 'CE-9', and avian 'neurothelin', 'HT7', or '5A11'. When heterologously expressed in HeLa cells, glycosylated immunoreactive protein was expressed at the plasma membrane. In the case of the endogenous protein in RC.SV3 cells, interferon-gamma and A23187 decreased, and fetal calf serum increased, steady-state mRNA levels. Thus, this molecule exhibits a high degree of cell type-specific expression in the kidney and undergoes regulation by cytokines and serum in kidney epithelial cells.

Amino Acid Sequence↗

PubMed1996

Characterization of oxalate transport by the human erythrocyte band 3 protein.

This paper describes characteristics of the transport of oxalate across the human erythrocyte membrane. Treatment of cells with low concentrations of H2DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) inhibits Cl(-)-Cl- and oxalate-oxalate exchange to the same extent, suggesting that band 3 is the major transport pathway for oxalate. The kinetics of oxalate and Cl- self-exchange fluxes indicate that the two ions compete for a common transport site; the apparent Cl- affinity is two to three times higher than that of oxalate. The net exchange of oxalate for Cl-, in either direction, is accompanied by a flux of H+ with oxalate, as is also true of net Cl(-)-SO4(2-) exchange. The transport of oxalate, however, is much faster than that of SO4(2-) or other divalent anions. Oxalate influx into Cl(-)-containing cells has an extracellular pH optimum of approximately 5.5 at 0 degrees C. At extracellular pH below 5.5 (neutral intracellular pH), net Cl(-)-oxalate exchange is nearly as fast as Cl(-)-Cl- exchange. The rapid Cl(-)-oxalate exchange at acid extracellular pH is not likely to be a consequence of Cl- exchange for monovalent oxalate (HOOC-COO-; pKa = 4.2) because monocarboxylates of similar structure exchange for Cl- much more slowly than does oxalate. The activation energy of Cl(-)-oxalate exchange is about 35 kCal/mol at temperatures between 0 and 15 degrees C; the rapid oxalate influx is therefore not a consequence of a low activation energy. The protein phosphatase inhibitor okadaic acid has no detectable effect on oxalate self-exchange, in contrast to a recent finding in another laboratory (Baggio, B., L. Bordin, G. Clari, G. Gambaro, and V. Moret. 1993. Biochim. Biophys. Acta. 1148:157-160.); our data provide no evidence for physiological regulation of anion exchange in red cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Permeability characteristics of erythrocyte membrane to okadaic acid and calyculin A.

The rates of transport of the protein phosphatase inhibitors okadaic acid and calyculin A through rabbit erythrocyte membranes have been estimated by measuring protein phosphatase type 2A (PP2A) activity in lysates. High concentrations of okadaic acid (100 nM) cause rapid (t 1/2 approximately 10 min) inhibition of PP2A. However, the t 1/2 for okadaic acid influx is much longer because the concentration is much higher than the concentration inhibiting 50% of the maximal response (IC50). The estimated t 1/2 is over 1 h at 37 degrees C and over 4 h at 25 degrees C. The effect of low extracellular pH indicates that the undissociated acid is the permeant species. It takes hours to reverse the effect of okadaic acid, because the efflux must proceed through several half times before the concentration is below the IC50 for PP2A. The permeation of calyculin A in contrast to okadaic acid is too fast to measure at 25 degrees C. Our results indicate that okadaic acid entry into erythrocytes is slower than is generally believed; it is crucial to consider concentration, temperature, pH, and time of exposure to okadaic acid to interpret the effects of this agent on intact cells.

Animals↗

PubMed1995

Anion exchange protein in Southeast Asian ovalocytes: heterodimer formation between normal and variant subunits.

Chemical cross-linking has been used to determine the composition of the erythrocyte band 3 protein dimer in Southeast Asian ovalocytes (SAO). Individuals with SAO are heterozygous for a mutation in which residues 400-408 of band 3 are deleted. Normal and variant protein are present in equal amounts, but the SAO protein does not transport anions or bind stilbenedisulfonates with high affinity. We find that the rate constant for 35SO4(2-) efflux from SAO cells is about 50% that of normal cells, but the time course is a single exponential, indicating that there is no detectable heterogeneity in the distribution of SAO band 3 in the population of cells. Treatment of intact cells with the homobifunctional crosslinker BS3 (bis[sulfosuccinimido]suberate) produces similar amounts of covalent dimer in both normal and SAO cells. In SAO cells, copies of normal band 3 can be distinguished from SAO band 3 by treating with H2DIDS to form a crosslink between major chymotryptic fragments (60 kDa and 35 kDa) within one subunit. Successive treatment of cells with [3H]-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate ([3H]H2DIDS), BS3, and chymotrypsin gives 3H-labeled products that include homodimer of normal band 3 as well as products of crosslinking normal band 3 with the 60- and 35-kDa fragment of SAO band 3. The results are in semiquantitative agreement with a model in which covalent dimer forms between normal and SAO subunits with the same probability as between two normal subunits. These results indicate that the normal copy of band 3, complexed in a heterodimer with SAO band 3, reacts with H2DIDS as in normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes.

One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na-gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1995

Renal chloride-bicarbonate exchangers.

This review discusses the molecular identities and functional properties of sodium-independent Cl(-)-HCO3- exchangers in the kidney. The main sites of renal Cl(-)-HCO3- exchange are in the intercalated cells of the collecting duct. The function of Cl(-)-HCO3- exchange is to provide a pathway for base efflux to balance the ATP-driven H+ efflux in cells that carry out transcellular net transport of H+. In the alpha-intercalated cell, which secretes H+ into the lumen, there is now excellent evidence that the basolateral Cl(-)-HCO3- exchanger is an N-terminal truncated form of the erythrocyte anion exchanger 1 (band 3) protein. In the beta-intercalated cell, which secretes HCO3-, it is well established that there is a Cl(-)-HCO3- exchanger in the apical membrane. Functional, immunocytochemical, and biochemical evidence indicate that the apical Cl(-)-HCO3- exchanger is not a product of the anion exchanger 1 gene. The identity of this protein remains uncertain.

Animals↗

PubMed1995

Transitional differentiation patterns of principal and intercalated cells during renal collecting duct development.

The developing renal collecting duct epithelium of neonatal rabbits exhibits 3 different zones. The ampullary tip epithelium acts as an embryonic inducer and is responsible for the generation of all of the nephron anlagen. It pilots the whole microarchitecture of the kidney. In the ampullary neck epithelium multiple cell divisions cause the elongation of the embryonic collecting duct so that the organ can grow. Finally, the cells in the ampullar shaft transdifferentiate into the functional collecting duct epithelium (CD) consisting of Principal (P) and various kinds of Intercalated (IC) cells. It is unknown by which morphogenic mechanisms the ampullar cells develop into the heterogeneously composed collecting duct epithelium. Using both morphological and immunohistochemical methods, we investigated the transdifferentiation patterns leading from the ampullar epithelium to the P and IC cells in the neonatal kidney. An electron microscope analysis of the cortico-medullary course of the developing collecting duct revealed that conspicuous morphological alterations start in the neck of the ampulla. The lumen of the neck region is narrowed to a slit. While most of the cells in the ampullar tip exhibit few, short microvilli, the neck cells bear numerous, extremely long microvilli at their apical cell poles. All of the neck cells exhibit the same cytoplasmic staining pattern and the same number of mitochondria. Farther down in the shaft, clearly recognizable P and IC cells are found. Thus, differentiation into P and IC cells starts with a transitional precursor cell type in the ampullar neck. Perfusion culture experiments with the embryonic collecting duct epithelium made it possible to generate transitional and differentiated cell types for the first time under in vitro conditions. The cultured epithelial cells showed characteristics common to both P and IC cells. Immunohistochemical findings revealed that morphological differentiation starts before the functional properties of P and IC cells can be detected.

Animals↗

PubMed1994

Red blood cell band 3. Lysine 539 and lysine 851 react with the same H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid) molecule.

The band 3 protein of the red blood cell membrane catalyzes anion exchange that is inhibited by the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid). There is one H2DIDS binding site per 95,000-Da band 3 polypeptide. The single bound H2DIDS molecule can react covalently with 2 different lysine residues. The 2 lysines that react covalently with H2DIDS have been localized directly by sequencing fragments of human band 3 from cells labeled with [3H]H2DIDS. The most rapid covalent reaction is with Lys-539, in agreement with site-directed mutagenesis studies. The slower reaction is with Lys-851, which is known to be the primary site of binding of another anion transport inhibitor, pyridoxal phosphate (Kawano et al., 1988). These results indicate that the protein is folded to bring these 2 residues into close enough proximity to react covalently with the same H2DIDS molecule. In addition to defining the residues that react with H2DIDS, these studies have also defined new in situ proteolytic cleavage sites in band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1.

We have examined inhibition of swelling-induced K-Cl cotransport in rabbit red blood cells by calyculin A, a potent serine-threonine protein phosphatase inhibitor, to determine whether transport is regulated by phosphatase type 1 or type 2A. Calyculin A blocks K(Rb) influx [half-maximal inhibitory concentration (IC50) = 3-6 nM] 10 times more potently than a second phosphatase inhibitor, okadaic acid (IC50 = 40 nM), consistent with earlier pharmacological studies showing that calyculin A inhibits phosphatase type 1 10 times more effectively than does okadaic acid. Calyculin A always inhibits Rb influx when added either before or after cell swelling, indicating that the phosphatase must operate continually to first activate and then maintain high transport rates in swollen cells. Similarly, N-ethylmaleimide (NEM) fails to stimulate K-Cl cotransport only when added to cells pretreated with calyculin A. Therefore, like cell swelling, activation of K-Cl cotransport by NEM involves a phosphatase sensitive to calyculin A. We conclude that cell swelling and NEM activate K-Cl cotransport via a net dephosphorylation that appears to involve protein phosphatase type 1.

Animals↗

PubMed1992

Anion-proton cotransport through the human red blood cell band 3 protein. Role of glutamate 681.

The band 3 protein of the human red blood cell membrane contains a glutamate residue that must be protonated in order for divalent (SO4=) anion transport to take place at an appreciable rate. The carboxyl side chain on this glutamate residue can be converted to the primary alcohol by treatment of intact cells with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate) followed by reductive cleavage with BH4-. Edman degradation of CNBr fragments from band 3 labeled in intact cells with Woodward's reagent K and [3H]BH4- showed that Glu681 is heavily labeled under conditions in which Cl- exchange is inhibited, SO4= exchange is accelerated, and Cl- conductance is accelerated. No other glutamate residue in band 3 is detectably labeled under the conditions of these experiments, as demonstrated either by Edman degradation or by the lack of label in major known proteolytic fragments. It is concluded that Glu681 is the binding site for the H+ that is transported with SO4= during band 3-catalyzed H+/SO4= cotransport. This residue is conserved among all species of red cell band 3 (AE1) as well as the related proteins AE2 and AE3. Glu681 is the first amino acid residue in band 3 which has been identified as a binding site for a transported substrate (H+). The functional characteristics of this residue suggest that it lies within the transport pathway and can be alternately exposed to the intracellular and extracellular media.

Amino Acid Sequence↗

PubMed1991

A 45-kDa protein antigenically related to band 3 is selectively expressed in kidney mitochondria.

Anion exchange similar to that catalyzed by erythrocyte band 3 occurs across many nonerythroid cell membranes. To identify anion-exchange proteins structurally related to band 3, we immunoblotted rabbit kidney medullary membrane fractions with anti-band 3 antibodies. Immunoblots using antibodies to the cytoplasmic domain of band 3 revealed cross-reactive proteins in the plasma membrane fraction only. In contrast, two monoclonal antibodies against band 3 membrane domain labeled a 45-kDa protein; further immunoblotting and immunogold studies of membrane fractions and kidney sections using one of the anti-membrane domain antibodies showed that labeling was strongest in mitochondria of H(+)-secreting collecting duct cells. Tissue-to-tissue expression of the 45-kDa mitochondrial protein was variable: kidney medulla greater than heart greater than kidney cortex much greater than liver. We conclude that a 45-kDa protein with immunological cross-reactivity to the erythrocyte band 3 membrane domain is expressed in mitochondria in a highly cell-specific fashion and speculate that the protein may play a role in mitochondrial anion transport.

Animals↗

PubMed1991

Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide.

The mechanism of activation of KCl cotransport has been examined in rabbit red blood cells. Previous work has provided evidence that a net dephosphorylation is required for activation of transport by cell swelling. In the present study okadaic acid, an inhibitor of protein phosphatases, was used to test this idea in more detail. We find that okadaic acid strongly inhibits swelling-stimulated KCl cotransport. The IC50 for okadaic acid is approximately 40 nM, consistent with the involvement of type 1 protein phosphatase in transport activation. N-Ethylmaleimide (NEM) is well known to activate KCl cotransport in cells of normal volume. Okadaic acid, added before NEM, inhibits the activation of transport by NEM, indicating that a dephosphorylation is necessary for the NEM effect. Okadaic acid added after NEM inhibits transport only very slightly. After a brief exposure to NEM and rapid removal of unreacted NEM, KCl cotransport activates with a time delay that is similar to that for swelling activation. Okadaic acid causes a slight increase in the delay time. These findings are all consistent with the idea that NEM activates transport not by a direct action on the transport protein but by altering a phosphorylation-dephosphorylation cycle. The simplest hypothesis that is consistent with the data is that both cell swelling and NEM cause inhibition of a protein kinase. Kinase inhibition causes net dephosphorylation of some key substrate (not necessarily the transport protein); dephosphorylation of this substrate, probably by type 1 protein phosphatase, causes transport activation.

Animals↗

PubMed1990

Kinetics of activation and inactivation of swelling-stimulated K+/Cl- transport. The volume-sensitive parameter is the rate constant for inactivation.

Red blood cells of several species are known to exhibit a ouabain-insensitive, anion-dependent K+ (Rb+) flux that is stimulated by cell swelling. We have used rabbit red cells to study the kinetics of activation and inactivation of the flux upon step changes in tonicity. Sudden hypotonic swelling (210 mosmol) activates the flux after a lag period of 10 min at 37 degrees C and 30-50 min at 25 degrees C. In cells that were preswollen to activate the transporter, sudden shrinkage (by addition of hypertonic NaCl) causes a rapid inactivation of the flux; the time lag for inactivation is less than 2 min at 37 degrees C. A minimal model of the volume-sensitive KCl transport system requires two states of the transporter. The activated (A) state catalyzes transport at some finite rate (turnover number unknown because the number of transporters is unknown). The resting (R) state has a much lower or possibly zero transport rate. The interconversion between the states is characterized by unimolecular rate constants R k12 in equilibrium with k21 A. The rate of relaxation to any new steady state is equal to the sum of the rate constants k12 + k21. Because the rate of transport activation in a hypotonic medium is lower than the rate of inactivation in an isotonic medium, we conclude that the volume-sensitive rate process is inactivation (the A to R transition); that is, cell swelling activates transport by lowering k21. Three phosphatase inhibitors (fluoride, orthovanadate, and inorganic phosphate) all inhibit the swelling-activated flux and also slow down the rate of approach to the swollen steady state. This finding suggests that a net dephosphorylation is necessary for activation of the flux and that the net dephosphorylation takes place as a result of swelling-induced inhibition of a kinase rather than stimulation of a phosphatase.

Animals↗