RESUMO
Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.
Assuntos
Potássio , Simportadores de Sódio-Bicarbonato , Animais , Camundongos , Bicarbonatos/metabolismo , Sítios de Ligação , Antiportadores de Cloreto-Bicarbonato/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Cloretos/metabolismo , Transporte de Íons , Simulação de Dinâmica Molecular , Potássio/metabolismo , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.
Assuntos
Células Acinares/enzimologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glândulas Salivares/enzimologia , Animais , Células CHO , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/genética , Cricetulus , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Glândulas Salivares/citologia , Relação Estrutura-AtividadeRESUMO
TASK-2 (K2P5) was one of the earliest members of the K2P two-pore, four transmembrane domain K(+) channels to be identified. TASK-2 gating is controlled by changes in both extra- and intracellular pH through separate sensors: arginine 224 and lysine 245, located at the extra- and intracellular ends of transmembrane domain 4. TASK-2 is inhibited by a direct effect of CO2 and is regulated by and interacts with G protein subunits. TASK-2 takes part in regulatory adjustments and is a mediator in the chemoreception process in neurons of the retrotrapezoid nucleus where its pHi sensitivity could be important in regulating excitability and therefore signalling of the O2/CO2 status. Extracellular pH increases brought about by HCO3 (-) efflux from proximal tubule epithelial cells have been proposed to couple to TASK-2 activation to maintain electrochemical gradients favourable to HCO3 (-) reabsorption. We demonstrate that, as suspected previously, TASK-2 is expressed at the basolateral membrane of the same proximal tubule cells that express apical membrane Na(+)-H(+)-exchanger NHE-3 and basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-A, the main components of the HCO3 (-) transport machinery. We also discuss critically the mechanism by which TASK-2 is modulated and impacts the process of HCO3 (-) reclaim by the proximal tubule epithelium, concluding that more than a mere shift in extracellular pH is probably involved.
Assuntos
Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/fisiologia , Túbulos Renais Proximais/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Bicarbonatos/metabolismo , Humanos , Túbulos Renais Proximais/patologiaRESUMO
TASK-2 is a K2P K(+) channel considered as a candidate to mediate CO2 sensing in central chemosensory neurons in mouse. Neuroepithelial cells in zebrafish gills sense CO2 levels through an unidentified K2P K(+) channel. We have now obtained zfTASK-2 from zebrafish gill tissue that is 49 % identical to mTASK-2. Like its mouse equivalent, it is gated both by extra- and intracellular pH being activated by alkalinization and inhibited by acidification. The pHi dependence of zfTASK-2 is similar to that of mTASK-2, with pK 1/2 values of 7.9 and 8.0, respectively, but pHo dependence occurs with a pK 1/2 of 8.8 (8.0 for mTASK-2) in line with the relatively alkaline plasma pH found in fish. Increasing CO2 led to a rapid, concentration-dependent (IC50 ~1.5 % CO2) inhibition of mouse and zfTASK-2 that could be resolved into an inhibition by intracellular acidification and a CO2 effect independent of pHi change. Indeed a CO2 effect persisted despite using strongly buffered intracellular solutions abolishing any change in pHi, was present in TASK-2-K245A mutant insensitive to pHi, and also under carbonic anhydrase inhibition. The mechanism by which TASK-2 senses CO2 is unknown but requires the presence of the 245-273 stretch of amino acids in the C terminus that comprises numerous basic amino acids and is important in TASK-2 G protein subunit binding and regulation of the channel. The described CO2 effect might be of importance in the eventual roles played by TASK-2 in chemoreception in mouse and zebrafish.
Assuntos
Dióxido de Carbono/metabolismo , Neurônios/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Potenciais de Ação , Sequência de Aminoácidos , Animais , Dióxido de Carbono/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Brânquias/citologia , Brânquias/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Estrutura Terciária de Proteína , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
TASK-2 (K2P5.1) is a background K(+) channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gßγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-ß-S instead. Purified Gßγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-ß-S was present. The effects of GTP-γ-S and Gßγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257-K258 or K296-K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gß1 and Gß2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257-K258 (but not K296-K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-ß-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gßγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Sequência de Aminoácidos , Animais , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Tionucleotídeos/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Excitatory synaptic transmission stimulates brain tissue glycolysis. This phenomenon is the signal detected in FDG-PET imaging and, through enhanced lactate production, is also thought to contribute to the fMRI signal. Using a method based on Förster resonance energy transfer in mouse astrocytes, we have recently observed that a small rise in extracellular K(+) can stimulate glycolysis by >300% within seconds. The K(+) response was blocked by ouabain, but intracellular engagement of the Na(+)/K(+) ATPase pump with Na(+) was ineffective, suggesting that the canonical feedback regulatory pathway involving the Na(+) pump and ATP depletion is only permissive and that a second mechanism is involved. Because of their predominant K(+) permeability and high expression of the electrogenic Na(+)/HCO(3)(-) cotransporter NBCe1, astrocytes respond to a rise in extracellular K(+) with plasma membrane depolarization and intracellular alkalinization. In the present article, we show that a fast glycolytic response can be elicited independently of K(+) by plasma membrane depolarization or by intracellular alkalinization. The glycolytic response to K(+) was absent in astrocytes from NBCe1 null mice (Slc4a4) and was blocked by functional or pharmacological inhibition of the NBCe1. Hippocampal neurons acquired K(+)-sensitive glycolysis upon heterologous NBCe1 expression. The phenomenon could also be reconstituted in HEK293 cells by coexpression of the NBCe1 and a constitutively open K(+) channel. We conclude that the NBCe1 is a key element in a feedforward mechanism linking excitatory synaptic transmission to fast modulation of glycolysis in astrocytes.
Assuntos
Astrócitos/metabolismo , Espaço Extracelular/metabolismo , Glicólise/fisiologia , Potássio/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Fatores de TempoRESUMO
TASK-2 (KCNK5 or K(2P)5.1) is a background K(+) channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH(o)) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH(i)) is independent from that under the command of pH(o). It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH(o) had normal pH(i)-dependent gating. Increasing extracellular K(+) concentration acid shifts pH(o) activity curve of TASK-2 yet did not affect pH(i) gating of TASK-2. pH(o) modulation of TASK-2 is voltage-dependent, whereas pH(i) gating was not altered by membrane potential. These results suggest that pH(o), which controls a selectivity filter external gate, and pH(i) act at different gating processes to open and close TASK-2 channels. We speculate that pH(i) regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys(245)) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH(i). We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH(i)-dependence curve of TASK-2 as expected from its lower pK(a). We conclude that intracellular pH, together with pH(o), is a critical determinant of TASK-2 activity and therefore of its physiological function.
Assuntos
Regulação da Expressão Gênica , Túbulos Renais/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Sequência de Aminoácidos , Animais , Eletrofisiologia/métodos , Humanos , Concentração de Íons de Hidrogênio , Lisina/química , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Potássio/química , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Sulfatos/químicaRESUMO
The Cl- channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl- efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-mu1B, but not in LLC-PK1-mu1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl- channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.