RESUMO
Sweat Na(+) concentration ([Na(+)]) varies greatly among individuals and is particularly high in cystic fibrosis (CF). The purpose of this study was to determine whether excess sweat [Na(+)] differentially impacts thirst drive and other physiological responses during progressive dehydration via exercise in the heat. Healthy subjects with high-sweat [Na(+)] (SS) (91.0 ± 17.3 mmol/l), Controls with average sweat [Na(+)] (43.7 ± 9.9 mmol/l), and physically active CF patients with very high sweat [Na(+)] (132.6 ± 6.4 mmol/l) cycled in the heat without drinking until 3% dehydration. Serum osmolality increased less (P < 0.05) in CF (6.1 ± 4.3 mosmol/kgH(2)O) and SS (8.4 ± 3.0 mosmol/kgH(2)O) compared with Control (14.8 ± 3.5 mosmol/kgH(2)O). Relative change in plasma volume was greater (P < 0.05) in CF (-19.3 ± 4.5%) and SS (-18.8 ± 3.1%) compared with Control (-14.3 ± 2.3%). Thirst during exercise and changes in plasma levels of vasopressin, angiotensin II, and aldosterone relative to percent dehydration were not different among groups. However, ad libitum fluid replacement was 40% less, and serum NaCl concentration was lower for CF compared with SS and Control during recovery. Despite large variability in sweat electrolyte loss, thirst appears to be appropriately maintained during exercise in the heat as a linear function of dehydration, with relative contributions from hyperosmotic and hypovolemic stimuli dependent upon the magnitude of salt lost in sweat. CF exhibit lower ad libitum fluid restoration following dehydration, which may reflect physiological cues directed at preservation of salt balance over volume restoration.
Assuntos
Fibrose Cística/metabolismo , Exercício Físico/fisiologia , Temperatura Alta , Sódio/metabolismo , Suor/metabolismo , Sede/fisiologia , Adulto , Aldosterona/sangue , Angiotensina II/sangue , Peso Corporal/fisiologia , Estudos de Casos e Controles , Fibrose Cística/fisiopatologia , Desidratação/fisiopatologia , Feminino , Humanos , Masculino , Concentração Osmolar , Volume Plasmático/fisiologia , Vasopressinas/sangueRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is both a member of the ATP-binding cassette superfamily and a Cl(-)-selective ion channel. We investigated the permeation pathway of human CFTR with measurements on conduction and open-channel blockade by diphenylamine-2-carboxylic acid (DPC). We used site-directed mutagenesis and oocyte expression to locate residues in transmembrane domain (TM) 6 and TM 12 that contact DPC and control rectification and single-channel conductances. Thus, TM 12 and the previously investigated TM 6 line the CFTR pore. In each TM, residues in contact with DPC are separated by two turns of an alpha helix. The contributions of TM 6 and TM 12 to DPC block and Cl- permeation, however, are not equivalent. The resulting structural model for the conduction pathway may guide future studies of permeation in other Cl- channels and ATP-binding cassette transporters.
Assuntos
Canais Iônicos/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Condutividade Elétrica , Espaço Extracelular/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Permeabilidade , Ratos , ortoaminobenzoatos/metabolismo , ortoaminobenzoatos/farmacologiaRESUMO
Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR's regulatory "R" domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.
Assuntos
Proteínas de Bactérias/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Esfingomielina Fosfodiesterase/farmacologia , Animais , Proteínas de Bactérias/química , Catálise , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação Enzimática , Humanos , Potenciais da Membrana , Modelos Moleculares , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ligação Proteica , Conformação Proteica , Esfingomielina Fosfodiesterase/química , Staphylococcus aureus/enzimologiaRESUMO
The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.
Assuntos
Cloretos/metabolismo , Fibrose Cística/metabolismo , Ácido Flufenâmico/farmacologia , Proteínas de Membrana/metabolismo , ortoaminobenzoatos/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Canais de Cloreto , AMP Cíclico/metabolismo , Fibrose Cística/genética , Eletrofisiologia , Espaço Extracelular/metabolismo , Feminino , Indicadores e Reagentes , Cinética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , XenopusRESUMO
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Metanossulfonato de Etila/análogos & derivados , Animais , Ânions/metabolismo , Arginina/genética , Cisteína/genética , Cisteína/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dissulfetos/metabolismo , Condutividade Elétrica , Metanossulfonato de Etila/farmacologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lisina/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Mercaptoetanol/farmacologia , Mesilatos/farmacologia , Modelos Biológicos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Perfusão , XenopusRESUMO
ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.
Assuntos
Canais de Cloreto/antagonistas & inibidores , Peptídeos/fisiologia , Venenos de Escorpião/farmacologia , Animais , Canais de Cloro CLC-2 , Células Cultivadas , Canais de Cloreto/biossíntese , Canais de Cloreto/genética , Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Peptídeos/química , Coelhos , Venenos de Escorpião/química , Escorpiões/química , Escorpiões/fisiologia , XenopusRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) protein forms a Cl(-) channel found in the plasma membranes of many epithelial cells, including those of the kidney, gut and conducting airways. Mutation of the gene encoding CFTR is the primary defect in cystic fibrosis, a disease that affects approximately 30 000 individuals in the United States alone. Alteration of CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease. The basic mechanisms of permeation in this channel are not well understood. It is not known which portions of the protein contribute to forming the pore or which amino acid residues in those domains are involved in the biophysical processes of ion permeation. In this review, I will discuss (i) the present understanding of ion transport processes in the wild-type CFTR channel, (ii) the experimental approaches currently being applied to investigate the pore, and (iii) a proposed structure that takes into account the present data on mechanisms of ion selectivity in the CFTR channel and on blockade of the pore by open-channel blockers.
Assuntos
Permeabilidade da Membrana Celular , Canais de Cloreto , Regulador de Condutância Transmembrana em Fibrose Cística , Sequência de Aminoácidos , Animais , Canais de Cloreto/química , Canais de Cloreto/fisiologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência MolecularRESUMO
The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Ativação do Canal Iônico/fisiologia , Alanina/genética , Substituição de Aminoácidos , Animais , Ânions/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Mutagênese/fisiologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Água/química , XenopusRESUMO
The mechanism of Ca(2+)-dependent control of hypotonic cell volume regulation was investigated in the isolated, nonperfused renal proximal straight tubule. When proximal tubules were exposed to hypotonic solution with 1 mM Ca2+, cells swelled rapidly and then underwent regulatory volume decrease (RVD). This treatment resulted in an increase in intracellular free calcium concentration ([Ca2+]i) by a mechanism that had two phases: the first was a transient increase from baseline (136 nM) to a peak (413 nM) that occurred in the first 15-20 sec, but was followed by a rapid decay toward the pre-swelling levels. The second phase was characterized by a sustained elevation of [Ca2+]i above the baseline (269 nM), which was maintained over several minutes. The dependence of these two phases on extracellular Ca2+ was determined. Reduction of bath [Ca2+] to 10 or 1 microM partially diminished the transient phase, but abolished the sustained phase completely, such that [Ca2+]i fell below the baseline levels during RVD. It was concluded that the transient increase resulted predominantly from swelling-activated release of intracellular Ca2+ stores and that the sustained phase was due to swelling-activated Ca2+ entry across the plasma membrane. Ca2+ entry probably also contributed to the transient increase in [Ca2+]i. The time dependence of swelling-activated Ca2+ entry was also investigated, since it was previously shown that RVD was characterized by a "calcium window" period (less than 60 sec), during which extracellular Ca2+ was required. Outside of this time period, RVD would inactivate and could not be reactivated by subsequent addition of Ca2+. It was found that the Ca2+ permeability did not inactivate over several minutes, indicating that the temporal dependence of RVD on extracellular Ca2+ is not due to the transient activation of a Ca2+ entry pathway.
Assuntos
Cálcio/farmacologia , Túbulos Renais Proximais/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/farmacocinética , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Feminino , Soluções Hipotônicas , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Ionomicina/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Coelhos , Sódio/farmacocinética , Fatores de TempoRESUMO
The Ca2+ entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca2+] from 1 mM to 1 microM caused intracellular free calcium concentration ([Ca2+]i) to fall close to zero. Treatment with 10 microM verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca2+ also induced fluctuations in cell volume. However, isotonic treatment with 10 microM nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca2+]i or cell volume, indicating that the endogenous Ca2+ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca2+, they swelled and underwent normal RVD while [Ca2+]i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991. J. Membrane Biol. 123:149-160). When cells were swollen in the presence of verapamil or low bath [Ca2+], RVD was abolished and [Ca2+]i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca2+]i were abolished, but [Ca2+]i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca2+ entry but that Ca2+ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca2+ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca2+ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive "baseline" Ca2+ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca2+ entry pathway, which is responsible for the supply of Ca ions to the Ca(2+)-dependent mechanism by which cell volume regulation is achieved.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Túbulos Renais Proximais/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/farmacocinética , Canais de Cálcio/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Soluções Hipotônicas , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/fisiologia , Microscopia de Fluorescência , Nifedipino/farmacologia , Coelhos , Verapamil/farmacologiaRESUMO
The mechanism underlying the activation of hypotonic cell volume regulation was studied in rabbit proximal straight tubule (PST). When isolated non-perfused tubules were exposed to hypotonic solution, cells swelled rapidly and then underwent a regulatory volume decrease (RVD). The extent of regulation after swelling was highly dependent on extracellular Ca concentration ([Ca2+]o), with a half-maximal inhibition (K1/2) for [Ca2+]o of approximately 100 microM. RVD was blocked by the Ca-channel blockers verapamil, lanthanum, and the dihydropyridines (DHP) nifedipine and nitrendipine, implicating voltage-activated Ca channels in the RVD response. Using the fura-2 fluorescence-ratio technique, we observed that cell swelling caused a sustained rise in intracellular Ca ([Ca2+]i) only when [Ca2+]o was normal (1 mM) but not when [Ca2+]o was low (1-10 microM). Furthermore, external Ca was required early on during swelling to induce RVD. If RVD was initially blocked by reducing [Ca2+]o or by addition of verapamil during hypotonic swelling, volume regulation could only be restored by subsequently inducing Ca entry within the first 1 min or less of exposure to hypotonic solution. These data indicate a "calcium window" of less than 1 min, during which RVD is sensitive to Ca, and that part of the Ca-dependent mechanism responsible for achieving RVD undergoes inactivation after swelling. It is concluded that RVD in rabbit PST is modulated by Ca via a DHP-sensitive mechanism in a time-dependent manner.
Assuntos
Cálcio/fisiologia , Di-Hidropiridinas/farmacologia , Túbulos Renais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Feminino , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Lantânio/farmacologia , Coelhos , Fatores de TempoRESUMO
It is evident from the present analysis that although a role for Ca2+ in controlling hypertonic cell volume regulation and RVI mechanisms has not been shown, Ca2+ plays a central role in activating and controlling hypotonic cell volume regulation and RVD mechanisms in most cells. However, this Ca2+ dependency is highly variable among cell types and tissues. Cells can be grouped into three general categories based on the relative dependency of RVD on Ca2+: 1) cells that are highly dependent on extracellular Ca2+ and the activation of Ca2+ influx, supposedly reflecting activation of Ca2+ channels, such as observed for the renal PST cells and osteosarcoma cells; 2) cells that are not dependent on extracellular Ca2+ and Ca2+ influx but that require at least a certain basal intracellular Ca2+ level or transient release of Ca2+ from internal stores, such as observed for the Ehrlich ascites tumor cells and medullary thick ascending limb cells; and 3) cells that display little if any Ca2+ dependency, such as the lymphocytes. There is initial evidence that this variable dependency of RVD on Ca2+ may reflect, in large part, a variable Ca2+ threshold of RVD processes, although this notion has not been fully investigated. The site and mechanism of Ca2+ dependency of RVD are poorly understood. Initial studies pointed to a possible direct control of K+ and/or Cl- channels by Ca2+ to modulate KCl efflux and, hence, RVD. This view appears to be too simplistic, however, as it is increasingly evident that the ion channels involved in RVD may not be directly Ca2+ dependent and that some other regulatory process controlling the channels, perhaps a phosphorylation step, may be the Ca(2+)-dependent event. Given the added complexity of the time-dependent variability of the action of Ca2+, i.e., the Ca2+ window, coupled with the variability of the RVD mechanisms among cell and tissue types, it is likely that the RVD mechanism is a highly complex process involving events and biochemical pathways throughout the cell rather than events simply localized to the inner face of the plasma membrane. It remains for future studies to determine the exact biochemical events that underly the RVD mechanism and its control, and the Ca2+ dependency of each step, before a full understanding will be attained of the role of Ca2+ in modulating RVD.
Assuntos
Cálcio/fisiologia , Tamanho Celular/fisiologia , Animais , Transporte Biológico , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Glibureto/farmacologia , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Oócitos/fisiologia , Xenopus/fisiologia , Animais , Feminino , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Técnicas de Patch-ClampRESUMO
Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cloreto/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Nitrobenzoatos/farmacologia , ortoaminobenzoatos/farmacologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Eletrofisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , XenopusRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel with distinctive kinetics. At the whole-cell level, CFTR currents in response to voltage steps are time independent for wild type and for the many mutants reported so far. Single channels open for periods lasting up to tens of seconds; the openings are interrupted by brief closures at hyperpolarized, but not depolarized, potentials. Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. In addition, the open-channel burst duration is decreased 10-fold compared with wild-type channels. S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Selectivity of the channel to a range of anions is also affected by this mutation. Furthermore, the permeation properties change during the relaxations, which suggests that there is an interaction between gating and permeation in this mutant. The existence of a mutation that confers voltage dependence upon CFTR currents and that changes kinetics and permeation properties of the channel suggests a functional role for the 11th transmembrane domain in the pore in the wild-type channel.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação do Canal Iônico/fisiologia , Estrutura Terciária de Proteína/genética , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Ativação do Canal Iônico/genética , Magnésio/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Permeabilidade/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Xenopus , ortoaminobenzoatos/farmacologiaRESUMO
Blockade of the CFTR chloride channel by glibenclamide was studied in Xenopus oocytes using two-electrode voltage-clamp recordings, macropatch recordings, and summations of single-channel currents, in order to test a kinetic model recently developed by us from single-channel experiments. Both the forward and reverse macroscopic reactions, at negative and positive membrane potential V(M), respectively, were slow in comparison to those reactions for other CFTR pore blockers such as DPC and NPPB, resulting in prominent relaxations on the order of tens of milliseconds. The rate of the reverse reaction was voltage-dependent, and dependent on the Cl(-) driving force, while that of the forward reaction was not. In inside-out macropatches, block and relief from block occurred in two distinct phases that differed in apparent affinity. The results are consistent with the presence of multiple glibenclamide binding sites in CFTR, with varying affinity and voltage dependence; they support the kinetic model and suggest experimental approaches for identification of those sites by mutagenesis.
Assuntos
Antiarrítmicos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Glibureto/farmacologia , Animais , Antiarrítmicos/metabolismo , Sítios de Ligação , Glibureto/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Oócitos , Técnicas de Patch-Clamp , Ligação Proteica , XenopusRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.
Assuntos
AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Feminino , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Humanos , Técnicas In Vitro , Proteínas de Membrana/genética , Oócitos/metabolismo , RNA Complementar/genética , RNA Mensageiro/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Serotonina/metabolismo , Receptores 5-HT1 de Serotonina , Xenopus laevisRESUMO
Patients with cystic fibrosis (CF) display defects in airway ion transport, but the influence of airway transport phenotype on improved prognosis is not known. We studied airway bioelectric properties in five CF patients with the rare A455E mutation that is associated with mild pulmonary disease. We also evaluated five patients possessing premature truncation mutations (G542X and R553X) for which an association with mild pulmonary disease has not been as well established. We found no evidence in vivo that a mild lung disease mutation in the CF transmembrane regulator gene (CFTR) led to correction or partial correction of: (1) unstimulated Cl- secretion; (2) beta-agonist-activated Cl- secretion; (3) basal sodium reabsorption; or (4) amiloride-sensitive airway sodium transport. Early phase therapeutic trials in CF, including human gene transfer trials, rely heavily on improvements in airway potential difference to identify promising interventions and an improved prognosis. Based on our findings in a naturally occurring group of CF patients with an improved pulmonary prognosis (A455E), one can argue that marked clinical benefit might be possible without any improvement whatsoever in airway bioelectric phenotype. Moreover, if genetic modifiers exist that influence the severity of a particular CFTR mutation (e.g., A455E), these may be independent of human airway Cl-secretion in vivo, since we detected minimal Cl--secretory responses in patients with A455E.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Transporte de Íons , Mutação , Mucosa Nasal/metabolismo , Adolescente , Agonistas Adrenérgicos beta/farmacologia , Adulto , Amilorida/farmacologia , Animais , Células COS/metabolismo , Criança , Cloretos/metabolismo , AMP Cíclico/farmacologia , Fibrose Cística/fisiopatologia , Feminino , Genótipo , Humanos , Transporte de Íons/efeitos dos fármacos , Isoproterenol/farmacologia , Masculino , Potenciais da Membrana , Mucosa Nasal/fisiopatologia , Prognóstico , Sódio/metabolismoRESUMO
Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.