RESUMO
Two electroneutral, Na+-driven HCO3- transporters, the Na+-driven Cl-/HCO3- exchanger and the electroneutral Na+/HCO3- cotransporter, have crucial roles in regulating intracellular pH in a variety of cells, including cardiac myocytes, vascular smooth-muscle, neurons and fibroblasts; however, it is difficult to distinguish their Cl- dependence in mammalian cells. Here we report the cloning of three variants of an electroneutral Na+/HCO3- cotransporter, NBCn1, from rat smooth muscle. They are 89-92% identical to a human skeletal muscle clone, 55-57% identical to the electrogenic NBCs and 33-43% identical to the anion exchangers. When expressed in Xenopus oocytes, NBCn1-B (which encodes 1,218 amino acids) is electroneutral, Na+-dependent and HCO3(-)-dependent, but not Cl(-)-dependent. Oocytes injected with low levels of NBCn1-B complementary RNA exhibit a Na+ conductance that 4,4-diisothiocyanatostilbene-2,2'-disulphonate stimulates slowly and irreversibly.
Assuntos
Bicarbonatos , Proteínas de Transporte/fisiologia , Canais de Sódio/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Transporte Biológico , Cloretos/metabolismo , Clonagem de Organismos , DNA Complementar , Eletrofisiologia , Humanos , Dados de Sequência Molecular , RNA Complementar , Ratos , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , Canais de Sódio/genética , Simportadores de Sódio-Bicarbonato , XenopusRESUMO
Electrogenic cotransport of Na+ and HCO-3 is a crucial element of HCO-3 reabsorption in the renal proximal tubule (PT). An electrogenic Na+-HCO-3 cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928-1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a approximately 130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl-/HCO-3 exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at approximately 130 kDa in both rat and rabbit, as well as a single approximately 160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-micrometer cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.
Assuntos
Proteínas de Transporte/metabolismo , Rim/metabolismo , Ambystoma , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular , Eletrofisiologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting , Imuno-Histoquímica , Coelhos , Ratos , Ratos Sprague-Dawley , Simportadores de Sódio-BicarbonatoRESUMO
The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl- absorption linked to Na+ were investigated in isolated perfused tubules using Cl--selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl- (aiCl) is 11.3 +/- 0.5 mm, the basolateral (V1), apical (V2), and transepithelial (V3) potential differences are -68 +/- 1.2 mV, +62 +/- 1.2 mV and -6.4 +/- 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, aiCl falls by 1.3 +/- 0.3 mm and V2 hyperpolarizes by +11.4 +/- 1.7 mV. Subsequent removal of Na+ from the lumen causes aiCl to fall further by 2.3 +/- 0.4 mm and V2 to hyperpolarize further by +15.3 +/- 2.4 mV. The contribution of transporters and channels to the observed changes of aiCl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on aiCl. The effects of luminal HCO-3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO-3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO3 exchange. Several lines of evidence favor conductive Cl- transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the aiCl by 2.4 +/- 0.6 mm or 2.9 +/- 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO-3-free Ringer, the equilibrium potential for Cl- does not differ from the membrane potential V2. Finally, the logarithmic changes in aiCl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl- channels at the apical and basolateral cell membranes of the proximal tubule.
Assuntos
Ambystoma/fisiologia , Membrana Celular/fisiologia , Canais de Cloreto/fisiologia , Cloretos/fisiologia , Animais , Transporte de Íons , Potenciais da Membrana , Sódio/fisiologiaRESUMO
The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Túbulos Renais Proximais/química , Canais de Potássio/fisiologia , Trifosfato de Adenosina/farmacologia , Ambystoma , Animais , Bário/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cátions/metabolismo , Células Cultivadas , Citidina Trifosfato/farmacologia , Diazóxido/farmacologia , Diuréticos , Condutividade Elétrica , Glibureto/farmacologia , Guanosina Trifosfato/farmacologia , Hipoglicemiantes/farmacologia , Inosina Trifosfato/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Cinética , Microvilosidades/química , Microvilosidades/enzimologia , Técnicas de Patch-Clamp , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Tálio , Nucleotídeos de Timina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Functional coupling of Na+,K+-ATPase pump activity to a basolateral membrane (BLM) K+ conductance is crucial for sustaining transport in the proximal tubule. Apical sodium entry stimulates pump activity, lowering cytosolic [ATP], which in turn disinhibits ATP-sensitive K+ (KATP) channels. Opening of these KATP channels mediates hyperpolarization of the BLM that facilitates Na+ reabsorption and K+ recycling required for continued Na+,K+-ATPase pump turnover. Despite its physiological importance, little is known about the regulation of this channel. The present study focuses on the regulation of the BLM KATP channel by second messengers and protein kinases using membrane patches from dissociated, polarized Ambystoma proximal tubule cells. The channel is regulated by protein kinases A and C, but in opposing directions. The channel is activated by forskolin in cell-attached (c/a) patches, and by PKA in inside-out (i/o) membrane patches. However, phosphorylation by PKA is not sufficient to prevent channel rundown. In contrast, the channel is inhibited by phorbol ester in c/a patches, and PKC decreases channel activity (nPo) in i/o patches. The channel is pH sensitive, and lowering cytosolic pH reduces nPo. Increasing intracellular [Ca2+] ([Ca2+]i) in c/a patches decreases nPo, and this effect is direct since [Ca2+]i inhibits nPo with a Ki of approximately 170 nM in i/o patches. Membrane stretch and hypotonic swelling do not significantly affect channel behavior, but the channel appears to be regulated by the actin cytoskeleton. Finally, the activity of this BLM KATP channel is coupled to transcellular transport. In c/a patches, maneuvers that inhibit turnover of the Na+,K+-ATPase pump reduce nPo, presumably due to a rise in intracellular [ATP], although the associated cell depolarization cannot be ruled out as the possible cause. Conversely, stimulation of transport (and thus pump turnover) leads to increases in nPo, presumably due to a fall in intracellular [ATP]. These results show that the inwardly rectifying KATP channel in the BLM of the proximal tubule is a key element in the feedback system that links cellular metabolism with transport activity. We conclude that coupling of this KATP channel to the activity of the Na+,K+-ATPase pump is a mechanism by which steady state NaCl reabsorption in the proximal tubule may be maintained.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Túbulos Renais Proximais/química , Canais de Potássio/fisiologia , Ambystoma , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/metabolismo , Cálcio/farmacologia , Membrana Celular/química , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Ativação Enzimática , Células Epiteliais/química , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Túbulos Renais Proximais/enzimologia , Técnicas de Patch-Clamp , Fosforilação , Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/farmacologiaRESUMO
The electrogenic Na/HCO3 cotransporter (symporter) is the major HCO3- transporter of the renal proximal tubule (PiT), located at the basolateral membrane (BLM), and also plays a noteworthy role in Na+ reabsorption. HCO3 transporters are important for regulation of intracellular pH (pHi) in most cells and also thereby regulate blood pH. This electrogenic Na/HCO3 cotransporter was first discovered using perfused Ambystoma tigrinum (salamander) renal, proximal tubules. This novel cotransporter mediates the movement of one Na+ ion with several HCO3- ions, making it electrogenic, is blocked by stilbene compounds, but does not depend on intra- or extracellular Cl-. This and similar cotransporters have been found in a number of tissues and cell types. Recently, we used Xenopus-laevis oocytes to expression clone the salamander renal electrogenic Na Bicarbonate Cotransporter (NBC). Using microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi), we followed oocyte expression after injecting poly (A)+, fractioned poly (A)+, or cRNA. All experimental solutions contained 100 microM ouabain to block the Na+/K+ pump. Our expression assay was to apply 1.5% CO2/10 mM HCO3- (pH 7.5), allow pHi to stabilize from the CO2-induced acidification, and then remove bath Na+. Removing bath Na+ from native oocytes and water-injected controls, hyperpolarized the oocytes by approximately 5 mV and had no effect on pHi. However, for oocytes injected with poly (A)+ RNA, removing Na+ transiently depolarized the cell by approximately 10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. Electrophoretic fractionation of the poly (A)+ RNA, enriched the expression signal. From the optimal expression-fraction, we constructed a size-selected cDNA library in pSPORT1. Screening our Ambystoma library yielded a single clone (aNBC). We could detect expression 3 days after injection of NBC cRNA. In aNBC-expressing oocytes, adding CO2/HCO3-elicited a large (> 50mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Na+ removal in CO2/HCO3-depolarized the cell by > 40mV and decreased pHi, aNBC encodes a protein of 1035 amino acids with several putative membrane-spanning domains, and has a low level of amino-acid homology (approximately 30% to the AE family of Cl-HCO3 exchangers. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters and, together with the AE family, defines a new superfamily of HCO3- transporters. Using aNBC to screen a rat-kidney cDNA library, we identified a full-length cDNA clone (rNBC), rNBC encodes a protein of 1035 amino acids, is 86% identical to aNBC, and can be functionally expressed in oocytes.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Proteínas de Transporte/fisiologia , Túbulos Renais Proximais/fisiologia , Equilíbrio Ácido-Base/genética , Animais , Proteínas de Transporte/genética , Clonagem Molecular , RNA Mensageiro/genética , Coelhos , Ratos , Simportadores de Sódio-Bicarbonato , Urodelos , Xenopus laevisRESUMO
The "black box" study of the passive and active electrical properties of single barriers in renal tubules has greatly contributed to the understanding of renal ion transport. With the advent of patch-clamp technology, it is feasible to record the ion flow through single channel proteins of the kidney, to infer their ion selectivity, gating properties, open pore conductivity, and to study directly the regulatory domains or sensors that control the gating of renal ion channels. Accordingly, it should be possible to upscale these microscopic parameters and predict the macroscopic membrane properties of renal membranes, using the knowledge of the microscopic single channel current (ij) for ion "j" under a physiologic driving force, the average number of channels in a single membrane patch (N), the open probability of a single channel (P(o)), the incidence of finding a channel in a population of membrane patches (f), and the area of a typical membrane patch (a). The experimental errors in the determination of each of these microscopic parameters are discussed. On the other hand, the macroscopic membrane properties of renal tubule cells cannot be reliably obtained from measurements of whole-cell patch-clamp, because of the polarized distribution of the dissipative electrical properties within a renal cell. The asymmetrical distribution of channel types, channel density and channel kinetics, between the apical and the basolateral membrane, precludes the use of the whole-cell conductance as the macroscopic reference. Instead, classical equivalent circuit analysis of renal epithelia is still necessary to obtain cell membrane parameters that validity represent the ensembles of single channels.
Assuntos
Canais Iônicos/fisiologia , Túbulos Renais/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Membrana Celular/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Técnicas de Patch-ClampRESUMO
Bicarbonate transporters are the principal regulators of pH in animal cells, and play a vital role in acid-base movement in the stomach, pancreas, intestine, kidney, reproductive system and central nervous system. The functional family of HCO3- transporters includes Cl- -HCO3- exchangers, three Na+/HCO3- cotransporters, a K+/HCO3- cotransporter, and a Na+-driven Cl- -HCO3- exchanger. Molecular information is sparse on HCO3- transporters, apart from Cl- -HCO3- exchangers ('anion exchangers'), whose complementary DNAs were cloned several years ago. Attempts to clone other HCO3- transporters, based on binding of inhibitors, protein purification or homology with anion exchangers, have so far been unsuccessful. Here we monitor the intracellular pH and membrane voltage in Xenopus oocytes to follow the expression of the most electrogenic transporter known: the renal 1:3 electrogenic Na+/HCO3- cotransporter from the salamander Ambystoma tigrinum. We now report the successful cloning and characterization of a cDNA encoding a cation-coupled HCO3- transporter. The encoded protein is 1,035 amino acids long with several potential membrane-spanning domains. We show that when it is expressed in Xenopus oocytes, this protein is electrogenic, Na+ and HCO3- dependent, and blocked by the anion-transport inhibitor DIDS, and conclude that it is the renal electrogenic sodium bicarbonate cotransporter (NBC).
Assuntos
Bicarbonatos/metabolismo , Proteínas de Transporte/genética , Rim/metabolismo , Sódio/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ambystoma , Sequência de Aminoácidos , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Clonagem Molecular , Eletrofisiologia , Expressão Gênica , Dados de Sequência Molecular , Oócitos , Simportadores de Sódio-Bicarbonato , Xenopus laevisRESUMO
The electrogenic Na+:HCO3- cotransporter (symporter) is the major transporter for HCO3- reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na+ reabsorption. We expression-cloned the salamander renal electrogenic Na+:Bicarbonate Cotransporter (NBC) in Xenopus laevis oocytes. After injecting poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi) All solutions contained ouabain to block the Na+/K+ pump (P-ATPase). After applying 1.5% CO2/10 mmol l-1 HCO3- (pH 7.5) and allowing pHi to stabilize from the CO2-induced acidification, we removed Na+. In native oocytes or water-injected controls, removing Na+ hyperpolarized the cell by -5 mV and had no effect on pHi. In oocytes injected with poly(A)+ RNA, removing Na+ transiently depolarized the cell by -10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. We enriched the signal by electrophoretic fractionation of the poly(A)+ RNA, and constructed a size-selected cDNA library in pSPORT1 using the optimal fraction. Screening the Ambystoma library yielded a single clone (aNBC). Expression was first obvious 3 days after injection of NBC cRNA. Adding CO2/HCO3- induced a large (> 50 mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Subsequent Na+ removal depolarized the cell by more than 40 mV and decreased pHi. aNBC is a full-length clone with a start Met and a poly(A)+ tail; it encodes a protein with 1025 amino acids and several putative membrane-spanning domains. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters. We used aNBC to screen a rat kidney cDNA library, and identified a full-length cDNA clone (rNBC) that encodes a protein of 1035 amino acids. rNBC is 86% identical to aNBC and can be functionally expressed in oocytes.
Assuntos
Ambystoma/metabolismo , Bicarbonatos/metabolismo , Proteínas de Transporte/fisiologia , Túbulos Renais Proximais/metabolismo , Sódio/metabolismo , Ambystoma/genética , Animais , Proteínas de Transporte/genética , Clonagem Molecular , DNA Complementar/genética , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Microinjeções , Oócitos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Simportadores de Sódio-Bicarbonato , Relação Estrutura-Atividade , Xenopus laevisRESUMO
Shaker genes encode voltage-gated potassium channels (Kv). We have shown previously that genes from Shaker subfamilies Kv1.1, 1.2, 1.4 are expressed in rabbit kidney. Recent functional and molecular evidence indicate that the predominant potassium conductance of the kidney medullary cell line GRB-PAP1 is composed of Shaker-like potassium channels. We now report the molecular cloning and functional expression of a new Shaker-related voltage-gated potassium channel, rabKv1.3, that is expressed in rabbit brain and kidney medulla. The protein, predicted to be 513 amino acids long, is most closely related to the Kv1.3 family although it differs significantly from other members of that family at the amino terminus. In Xenopus oocytes, rabKv1.3 cRNA expresses a voltage activated K current with kinetic characteristics similar to other members of the Kv1.3 family. However, unlike previously described Shaker channels, it is sensitive to glibenclamide and its single channel conductance saturates. This is the first report of the functional expression of a voltage-gated K channel clone expressed in kidney. We conclude that rabKv1.3 is a novel member of the Shaker superfamily that may play an important role in renal potassium transport.
Assuntos
Glibureto/farmacologia , Medula Renal/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , Primers do DNA , Feminino , Variação Genética , Biblioteca Genômica , Humanos , Canal de Potássio Kv1.3 , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Proteínas Associadas a Pancreatite , Filogenia , Reação em Cadeia da Polimerase , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Coelhos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The functional properties of an epithelium are inextricably linked to its polarized structure. It has been difficult to study polarity at the level of the single cell, since most epithelial cells lose their polarity within minutes after dissociation. We have developed a preparation of native, dissociated, Ambystoma proximal tubule cells that maintain structural and functional polarity for a minimum of 7 days in suspension. We have used these cells to explore cell surface polarity in a single cell. Electron microscope cytochemical and immunocytochemical studies show that alkaline phosphatase is localized exclusively to the apical brush border, whereas the Na(+)-K(+)-ATPase is restricted to the basolateral membrane. Just as in the proximal tubule in situ, a sharp structural transition between the apical and basolateral membrane domains is retained. The ZO-1 protein found at the tight junction in situ is not present on the membrane of the dissociated cells, but rather it is distributed in the cytoplasm. The actin cytoskeleton also remains polarized in the single cells, and its distribution and organization appear to help maintain cell polarity. Electrophysiological measurements show that these cells remain viable at least as long as they remain structurally polarized. Patch-clamp recordings from both the apical and basolateral membranes show that the distribution of several ion channel proteins maintains functional polarity. We hypothesize that, despite loss of the intercellular "gate" and membrane-associated ZO-1, the socalled "fence" function of the tight junctional complex is retained in these dissociated proximal tubule cells. This preparation may serve as a useful single cell model with which to study epithelial polarity and membrane trafficking pathways.
Assuntos
Polaridade Celular , Técnicas Citológicas , Túbulos Renais Proximais/fisiologia , Ambystoma , Animais , Membrana Celular/enzimologia , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Eletrofisiologia , Células Epiteliais , Epitélio/enzimologia , Epitélio/fisiologia , Canais Iônicos/fisiologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/enzimologia , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Fatores de Tempo , Proteína da Zônula de Oclusão-1RESUMO
Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.
Assuntos
GMP Cíclico/metabolismo , Ativação do Canal Iônico , Canais de Potássio/genética , Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Condutividade Elétrica , Biblioteca Gênica , Dados de Sequência Molecular , Oócitos/metabolismo , Filogenia , Canais de Potássio/classificação , Canais de Potássio/metabolismo , Biossíntese de Proteínas , Conformação Proteica , RNA Mensageiro/metabolismo , Coelhos , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , XenopusRESUMO
We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262-10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na+, K+, Rb+, Cs+, and Li+. It has a linear I-V relation with a conductance of 22.7 +/- 0.5 pS (n = 78) at room temperature. The Pcation/P(anion) ratio is about 60 and there is no measurable conductance for NMDG, Ca2+, Ba2+, and Mn2+. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10(-6) M and depolarization increases channel activity (NPo). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10(-4) M and 10(-3) M, ATP reduces NPo by 23% and 69%, respectively. Furthermore, since ADP (10(-3) M) reduces NPo by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a gamma-phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10(-4) M) or by cGMP-dependent protein kinase (10(-7) M) in the presence of 8-Br-cGMP (10(-5) M) and ATP (10(-4) M). The NSC channel is not sensitive to amiloride (10(-4) M cytoplasmic and/or extracellular) but flufenamic acid (10(-4) M) produces a voltage-dependent block, reducing NPo by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages. We conclude that the NCS channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.
Assuntos
Cálcio/fisiologia , Canais Iônicos/fisiologia , Córtex Renal/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular , Membrana Celular/fisiologia , Túbulos Renais Coletores/metabolismo , Camundongos , Nucleotídeos/farmacologia , Técnicas de Patch-ClampRESUMO
M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl-Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M-1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.
Assuntos
Túbulos Renais Coletores/fisiologia , Amilorida/farmacologia , Animais , Bário/farmacologia , Linhagem Celular , Cloretos/farmacologia , Condutividade Elétrica , Células Epiteliais , Epitélio/fisiologia , Córtex Renal , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Camundongos , Fatores de TempoRESUMO
We tested the effects of endothelin-1 (ET-1) on intracellular calcium concentration ([Ca2+]i) of cultured M-1 mouse cortical collecting duct cells. [Ca2+]i was measured using fura 2 and a fluorescent imaging system. At a concentration of extracellular calcium ([Ca2+]o) of 1 mM, ET-1 (10(-12) to 10(-7) M) increased [Ca2+]i. A second application of ET-1 had no effect on Ca2+. In contrast, application of arginine vasopressin after an initial exposure to ET-1 induced a second Ca2+ response. In the absence of extracellular Ca2+ (1 mM EGTA) ET-1 also elicited a Ca2+ peak, indicating participation of Ca2+ release from intracellular stores in the initial Ca2+ peak. At [Ca2+]o of 10 mM, ET-1 also induced an intracellular Ca2+ peak but [Ca2+]i remained significantly elevated. The Ca2+ plateau phase was abolished by nickel (10 or 100 microM) and nifedipine (0.1 or 1 microM). We conclude that ET-1 mediates an increase in [Ca2+]i by Ca2+ release from intracellular stores and activation of a nickel- and nifedipine-sensitive Ca2+ entry mechanism.
Assuntos
Cálcio/metabolismo , Endotelinas/farmacologia , Membranas Intracelulares/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Arginina Vasopressina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Espaço Extracelular/metabolismo , Córtex Renal , Túbulos Renais Coletores/citologia , Camundongos , Níquel/farmacologia , Nifedipino/farmacologia , Concentração OsmolarRESUMO
Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors.
Assuntos
GMP Cíclico/farmacologia , Canais Iônicos/genética , Canais Iônicos/fisiologia , Córtex Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Células Fotorreceptoras/fisiologia , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , DNA/genética , DNA/isolamento & purificação , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Poli A/genética , Poli A/isolamento & purificação , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
Modes of bicarbonate entry from tubule lumen to cell were examined in isolated Ambystoma proximal tubules, using determinations of transepithelial potential differences (V3). (1) Upon removal of luminal substrate, tubules first equilibrated in bilateral (lumen and bath) 94.72 mM Cl- and 10 mM HCO3- yielded a change in V3 between the experimental and control circumstances of +1.8 mV (delta V3). (2) The identical experiment conducted under the condition of symmetrical 4.72 mM Cl- produced a delta V3 of +7.6 mV. This reduction of luminal and bath Cl- generates an amplification of delta V3 by a factor of 4.4 and reflects a substantial increase in the paracellular Cl- shunt resistance. Ensuing experiments were conducted in bilateral nominally Cl(-)-free solutions and in the absence of luminal substrate. The experimental protocols are divided into several situations where HCO3- is removed from the lumen, bath, or lumen and bath; the HCO3- removal sequences are repeated in the presence of luminal SITS and then after SITS washout. 0.5 mM SITS (4-acetoamido-4-isothiocyanostilbene-2,2'-disulfonate) was applied exclusively to the luminal perfusate. (1) Removal of luminal HCO3- in the absence of SITS produces a delta V3 of -1.9 mV, whereas, in the presence of SITS, the delta V3 measures -1.3 mV. Subsequent removal of luminal HCO3- in the presence of bath HCO3- (in the presence of luminal SITS) yields a delta V3 of -1.0 mV. All of these measurements reflect a decrease in HCO3- current across the basolateral membrane Na+ (HCO3-)n co-transporter; the role of a possible Cl-/Anion- antiport cannot be assessed. (2) Removal of bath HCO3- in the absence of SITS yields a delta V3 of +1.5 mV, whereas, in the presence of SITS, the delta V3 value measures +1.2 mV. Subsequent removal of bath HCO3- in the absence of luminal HCO3- (in the presence of SITS) yields a delta V3 of +0.8 mV. These experiments are consistent with an increase in HCO3- current across the basolateral Na+(HCO3-)n co-transporter, do not rule out the possibility of an apical HCO3- conductance pathway, and diminish the likelihood of an apical Cl-/HCO3- antiport system.
Assuntos
Bicarbonatos/metabolismo , Túbulos Renais Proximais/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico , Ambystoma , Animais , Transporte Biológico Ativo , Cloretos/metabolismo , Epitélio/metabolismo , Feminino , Técnicas In Vitro , Potenciais da Membrana , PerfusãoRESUMO
Isosmotic transport of fluid across epithelial cell layers occurs by intraepithelial mechanisms that are not fully understood. Newer methods of measuring water flows across epithelia with higher resolution should now permit some key issues regarding solute-linked water transport to be clarified. Unstirred-layer effects are not likely to be serious sources of error in these measurements with judicious choice of experimental conditions. Progress in ultrastructural stereology has shown that in the proximal tubule most of the transporting membrane is located in the basal aspects of cells, making models based on a hyperosmolar lateral compartment less relevant. The current models of simple transcellular osmosis, though appealing for this simplicity, fail to account for some major experimental findings. Experimental design and methodological limitations have not yet achieved rigorous testing of whether or not epithelia can produce a perfectly isosmotic absorbate without any transepithelial driving forces. A better understanding of the mechanism of translocation of water through the lipid bilayer, the plasma membrane proteins, and special membrane structures like the tight junctions would significantly contribute to our knowledge of the mechanisms and intraepithelial routes by which water is transported by epithelia.
Assuntos
Epitélio/metabolismo , Água/metabolismo , Animais , Transporte Biológico Ativo , Permeabilidade da Membrana Celular , Concentração Osmolar , Coelhos , Ratos , Equilíbrio HidroeletrolíticoRESUMO
An electrophysiological approach is used to analyze the possible routes of osmotically driven water flow across the isolated perfused Ambystoma proximal tubule. The minimum hydraulic conductivities (Lp) of the cell membranes were estimated from the initial rate of change of intracellular activities of Na+ and K+ in response to a step gradient of 50 or 100 mosmol/kg sucrose. The Lp of the apical membrane is 1.30 X 10(-4) cm.s-1.osM-1 referred to the luminal epithelial surface and 2.45 X 10(-6) cm.s-1.osM-1 when corrected for amplification of the brush border (n = 8). The Lp of the basolateral membrane is 1.42 X 10(-4) cm.s-1.osM-1 referred to the basement membrane surface and 6.39 X 10(-6) cm.s-1.osM-1 when corrected for the amplification of the basal and lateral membranes (n = 5). Transepithelial water flows were generated in either direction by a unilateral step increase of osmolality with 100 mosmol sucrose. Bath-to-lumen flow increased paracellular transepithelial resistance (R3) by 48%; lumen-to-bath flow decreased R3 by only 3%. A bilateral increase in the osmolality of both solutions by 50 mosM had no significant effect on R3. Streaming potentials were observed during trans-epithelial water flow induced by unilateral gradients of sucrose; their polarity, magnitude, site of generation, and insensitivity to change of paracellular resistance are all indicative of water flow through paracellular structures, especially the lateral intercellular spaces. Contrary to earlier suggestions (J. M. Diamond, J. Membr. Biol. 51: 195-216, 1979), these potentials are not primarily diffusion potentials across anion-selective tight junctions resulting from solute polarization in the unstirred layers. Instead, a true electrokinetic basis for these streaming potentials is indicated by their continued presence after deletion of all Cl-. Thus water moves through both cellular and paracellular pathways in this epithelium.