RESUMEN
Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-dependent channel activity in only 1.5% of patches. With K(+) (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.
Asunto(s)
Canales de Calcio/metabolismo , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Empalme Alternativo/genética , Animales , Células COS , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio/química , Canales de Calcio/genética , Línea Celular , Electrofisiología , Receptores de Inositol 1,4,5-Trifosfato , Activación del Canal Iónico/efectos de los fármacos , Técnicas de Placa-Clamp , Probabilidad , Ratas , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/farmacocinética , Inositol 1,4,5-Trifosfato/metabolismo , Activación del Canal Iónico/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Canales de Calcio/química , Receptores de Inositol 1,4,5-Trifosfato , Activación del Canal Iónico/efectos de los fármacos , Isomerismo , Cinética , Modelos Biológicos , Oocitos/fisiología , Técnicas de Placa-Clamp , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.
Asunto(s)
Adenosina Trifosfato/metabolismo , Canales de Calcio , Activación del Canal Iónico/fisiología , Receptores Citoplasmáticos y Nucleares , Adenosina Trifosfato/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Sitios de Unión/fisiología , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/genética , Canales de Calcio/metabolismo , Citoplasma/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Oocitos/fisiología , Técnicas de Placa-Clamp , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , XenopusRESUMEN
The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.
Asunto(s)
Adenosina Trifosfato/farmacología , Adenosina/análogos & derivados , Adenosina/farmacología , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Adenosina/química , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión/fisiología , Agonistas de los Canales de Calcio/química , Canales de Calcio/química , Señalización del Calcio/fisiología , Citoplasma/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Activación del Canal Iónico/fisiología , Cinética , Ligandos , Oocitos/fisiología , Técnicas de Placa-Clamp , Receptores Citoplasmáticos y Nucleares/química , XenopusRESUMEN
We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)).
Asunto(s)
Canales de Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Iones , Secuencia de Aminoácidos , Animales , Ácido Aspártico/química , Sitios de Unión , Células COS , Calcio/metabolismo , Cationes , Membrana Celular/metabolismo , Electrofisiología , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Neuronas/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Valina/químicaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase- and ATP-regulated chloride channel, the activity of which determines the rate of electrolyte and fluid transport in a variety of epithelial tissues. Here we describe a mechanism that regulates CFTR channel activity, which is mediated by PDZ domains, a family of conserved protein-interaction modules. The Na(+)/H(+) exchanger regulatory factor (NHERF) binds to the cytoplasmic tail of CFTR through either of its two PDZ (PDZ1 and PDZ2) domains. A recombinant fragment of NHERF (PDZ1-2) containing the two PDZ domains increases the open probability (P(o)) of single CFTR channels in excised membrane patches from a lung submucosal gland cell line. Both PDZ domains are required for this functional effect, because peptides containing mutations in either domain are unable to increase channel P(o). The concentration dependence of the regulation by the bivalent PDZ1-2 domain is biphasic, i.e., activating at lower concentrations and inhibiting at higher concentrations. Furthermore, either PDZ domain alone or together is without effect on P(o), but either domain can competitively inhibit the PDZ1-2-mediated stimulation of CFTR. Our results support a molecular model in which bivalent NHERF PDZ domains regulate channel gating by crosslinking the C-terminal tails in a single dimeric CFTR channel, and the magnitude of this regulation is coupled to the stoichiometry of these interactions.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Membrana Celular/fisiología , Secuencia Conservada , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Activación del Canal Iónico , Cinética , Pulmón/fisiología , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Mucosa Respiratoria/fisiología , TransfecciónRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in Xenopus oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Fosforilación , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Testículo/metabolismo , TransfecciónRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Canales de Sodio/fisiología , Animales , Canales de Cloruro/fisiología , Femenino , Regulación de la Expresión Génica , Humanos , Activación del Canal Iónico , Transporte Iónico , Ratones , Oocitos , Transducción de Señal , XenopusRESUMEN
The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.
Asunto(s)
Canales de Calcio/metabolismo , Retículo Endoplásmico/química , Activación del Canal Iónico/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/fisiología , Membrana Celular/química , Membrana Celular/metabolismo , Conductividad Eléctrica , Estimulación Eléctrica , Retículo Endoplásmico/metabolismo , Expresión Génica/fisiología , Receptores de Inositol 1,4,5-Trifosfato , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Xenopus laevisRESUMEN
Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](i) from 500 +/- 50 nM in 0 [ATP](i) to 29 +/- 4 nM in 9.5 mM [ATP](i), with apparent ATP affinity = 0.27 +/- 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.
Asunto(s)
Adenosina Trifosfato/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Inositol 1,4,5-Trifosfato/metabolismo , Activación del Canal Iónico , Receptores Citoplasmáticos y Nucleares/metabolismo , Regulación Alostérica , Animales , Receptores de Inositol 1,4,5-Trifosfato , Oocitos , Técnicas de Placa-Clamp , Factores de Tiempo , XenopusRESUMEN
To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/patología , Portadores de Fármacos , Técnicas de Transferencia de Gen , Polilisina/administración & dosificación , Secuencia de Bases , Línea Celular Transformada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Cartilla de ADN , ADN Complementario , TransfecciónRESUMEN
Inositol 1,4,5-trisphosphate (IP3) [corrected] binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remained elevated (approximately 0.8) in the presence of saturating levels (10 microM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) approximately 2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 microM and Hill coefficient (Hinh) approximately 4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Retroalimentación , Femenino , Receptores de Inositol 1,4,5-Trifosfato , Activación del Canal Iónico , Cinética , Potenciales de la Membrana , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transducción de Señal , Xenopus laevisRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cloruros/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Conductividad Eléctrica , Femenino , Expresión Génica , Potenciales de la Membrana/fisiología , Oocitos/metabolismo , Xenopus laevisRESUMEN
The effects of Mg2+ and Ba2+ on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP3R) were studied by patch clamp of isolated nuclei from Xenopus oocytes. In 140 mM K+ the IP3R channel kinetics and presence of conductance substates were similar over a range (0-9.5 mM) of free Mg2+. In 0 mM Mg2+ the channel current-voltage (I-V) relation was linear with conductance of approximately 320 pS. Conductance varied slowly and continuously over a wide range (SD approximately 60 pS) and sometimes fluctuated during single openings. The presence of Mg2+ on either or both sides of the channel reduced the current (blocking constant approximately 0.6 mM in symmetrical Mg2+), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance approximately 120 pS near 0 mV and approximately 360 pS at +/-70 mV in symmetrical 2.5 mM Mg2+). Ba2+ exhibited similar effects on channel conductance. Mg2+ and Ba2+ permeated the channel with a ratio of permeability of Ba2+ to Mg2+ to K+ of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP3R.
Asunto(s)
Bario/farmacología , Canales de Calcio/fisiología , Cationes Bivalentes/farmacología , Magnesio/farmacología , Oocitos/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Conductividad Eléctrica , Femenino , Inositol 1,4,5-Trifosfato/fisiología , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Oocitos/efectos de los fármacos , Potasio/farmacología , Tiempo de Reacción , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Xenopus laevisRESUMEN
The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.
Asunto(s)
Canales de Sodio/metabolismo , Animales , Epitelio/metabolismo , Ratones , Mutagénesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Sodio/química , Canales de Sodio/genética , XenopusRESUMEN
Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.
Asunto(s)
Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Genes Reguladores/genética , Activación del Canal Iónico/fisiología , Animales , Canales de Cloruro/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Activación del Canal Iónico/genéticaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.
Asunto(s)
Adenosina Trifosfato/metabolismo , Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación del Canal Iónico , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Canales de Cloruro/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Perros , Hidrólisis , Riñón , Nucleótidos/metabolismo , Permeabilidad , FosforilaciónRESUMEN
Single-channel properties of the Xenopus inositol trisphosphate receptor (IP3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K+ as the charge carrier (cytoplasmic [IP3] = 10 microM, free [Ca2+] = 200 nM), the IP3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and approximately 300 pS at 60 mV. The channel was frequently observed with high open probability (mean P(o) = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants tau < 5 ms and approximately 20 ms; and at least three closed states with tau approximately 1 ms, approximately 10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and tau of the longest closed state. A novel "flicker" kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F1 and F2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F1 to F2 transitions. Channel run-down or inactivation (tau approximately 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (approximately 10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage-reversible inactivation. Mapping of functional channels indicates that the IP3R tends to aggregate into microscopic (<1 microm) as well as macroscopic (approximately 10 microm) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.
Asunto(s)
Canales de Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Oocitos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Electrofisiología , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Potenciales de la Membrana/fisiología , Oocitos/ultraestructura , Técnicas de Placa-Clamp , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , XenopusRESUMEN
Cystic fibrosis (CF) is characterized by abnormal regulation of epithelial ion and fluid transport due to mutations in the CF transmembrane conductance regulator (CFTR), an apical membrane-localized Cl- channel, that usually prevent it from exiting the endoplasmic reticulum. Defective or absent CFTR in the epithelium is believed to disrupt fluid balance in human airways and thereby contribute to chronic respiratory inflammation. Patch-clamp of the plasma membrane and outer membrane of the nuclear envelope of nuclei isolated from CFTR-expressing Chinese hamster ovary cells revealed that CFTR is associated with a regulated ATP channel in both membrane compartments. CFTR expression was also shown to be associated with permeability to another adenine nucleotide, adenosine 3'-phosphate 5'-phosphosulfate, the universal sulfate donor in cells. These results may provide a link between the ion channel function of CFTR and abnormal glycoprotein processing observed in CF.
Asunto(s)
Adenosina Trifosfato/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Retículo Endoplásmico/metabolismo , Fosfoadenosina Fosfosulfato/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Aparato de Golgi/metabolismo , Humanos , Potenciales de la Membrana , Proteínas Recombinantes/metabolismoRESUMEN
Cholinergic agonists stimulate isotonic fluid secretion in the parotid gland. This process is driven by the apical exit of Cl-, which enters the cells partly via Cl-/HCO-3 exchange across the basolateral membrane. Acidification of the cytosol by the extrusion of HCO-3 is prevented by the concomitant activation of the Na+/H+ exchanger (NHE), which is directly activated by cholinergic stimulation. Multiple isoforms of the NHE have been described in mammalian cells, but the particular isoform(s) present in salivary glands and their mechanism of activation have not been defined. Reverse transcriptase-polymerase chain reaction with isoform-specific primers was used to establish that NHE-1 and NHE-2, but not NHE-3 or NHE-4, are expressed in parotid glands. The presence of NHE-1 was confirmed by immunoblotting and immunofluorescence, which additionally demonstrated that this isoform is abundant in the basolateral membrane of acinar cells. The predominant role of NHE-1 in carbachol-induced Na+/H+ exchange was established pharmacologically using HOE694, an inhibitor with differential potency toward the individual isoforms. Because muscarinic agonists induce stimulation of protein kinases in acinar cells, we assessed the role of phosphorylation in the activation of the antiport. Immunoprecipitation experiments revealed that, although NHE-1 was phosphorylated in the resting state, no further phosphorylation occurred upon treatment with carbachol. Similar phosphopeptide patterns were observed in control and carbachol-treated samples. Together, these findings indicate that NHE-1, the predominant isoform of the antiporter in the basolateral membrane of acinar cells, is activated during muscarinic stimulation by a phosphorylation-independent event. Other processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of the antiporter, may be responsible for the activation of Na+/H+ exchange.