RESUMEN
The proposed Ca2+-activated Cl- channel protein Best1 (bestrophin 1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK (human embryonic kidney)-293 cells, and compared its properties with Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl- channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2-expressing cells had no detectable Ca2+-activated Cl- currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Δex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modelling of the Best1 protein structure.
Asunto(s)
Empalme Alternativo , Canales de Cloruro/genética , Proteínas del Ojo/genética , Astrocitos/metabolismo , Bestrofinas , Línea Celular Tumoral , Canales de Cloruro/metabolismo , Clonación Molecular , Exones , Proteínas del Ojo/metabolismo , Glioma/metabolismo , Células HEK293 , Humanos , Neuroglía/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
The Ca(2+) sensor stromal interacting molecule 1 (STIM1) and the Ca(2+) channel Orai1 mediate the ubiquitous store-operated Ca(2+) entry (SOCE) pathway activated by depletion of internal Ca(2+) stores and mediated through the highly Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca(2+) currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca(2+) imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Glioblastoma/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Potenciales de Acción , Astrocitos/metabolismo , Neoplasias Encefálicas/patología , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Humanos , Proteínas de la Membrana/genética , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula de Interacción Estromal 1 , Transcripción Genética , Regulación hacia ArribaRESUMEN
Store-operated calcium (Ca(2+)) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca(2+) sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca(2+) causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca(2+)-selective Ca(2+) release-activated Ca(2+) current (I(CRAC)). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca(2+) imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I(CRAC) in estrogen receptor-positive (ER(+)) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER(-)) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER(+) breast cancer cells represent the first example where the native SOCE pathway and I(CRAC) are mediated by Orai3. Future studies implicating Orai3 in ER(+) breast cancer progression might establish Orai3 as a selective target in therapy of ER(+) breast tumors.
Asunto(s)
Neoplasias de la Mama/metabolismo , Canales de Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptores de Estrógenos/metabolismo , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Técnicas de Placa-Clamp , Transporte de Proteínas , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Endotelio/patología , Regulación Enzimológica de la Expresión Génica , Trombina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Calcio/metabolismo , Células Endoteliales/citología , Endotelio/metabolismo , Endotelio Vascular/citología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Masculino , Modelos Biológicos , Isoformas de Proteínas , Ratas , Ratas Sprague-Dawley , Trombina/químicaRESUMEN
The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Glicoproteínas de Membrana/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Proteína ORAI1 , Técnicas de Placa-Clamp , ARN Interferente Pequeño/genética , Ratas , Molécula de Interacción Estromal 1 , Tapsigargina/farmacologíaRESUMEN
Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.
Asunto(s)
Canales de Calcio/fisiología , Calcio/fisiología , Endotelio Vascular/fisiología , Glicoproteínas de Membrana/fisiología , Canales Catiónicos TRPC/fisiología , Animales , Canales de Calcio/genética , División Celular , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Leucemia Basofílica Aguda/fisiopatología , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteína ORAI1 , Técnicas de Placa-Clamp , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Molécula de Interacción Estromal 1 , Tapsigargina/farmacología , Venas UmbilicalesRESUMEN
Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a D-[(3)H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypo-osmotic media stimulated Cl(-) currents and D-[(3)H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H(2)O(2) potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.
Asunto(s)
Aminoácidos Excitadores/metabolismo , Microglía/metabolismo , NADPH Oxidasas/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Zimosan/farmacología , Animales , Animales Recién Nacidos , Ácido Aspártico/metabolismo , Células Cultivadas , Encefalitis/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Microglía/efectos de los fármacos , NADPH Oxidasa 4 , NADPH Oxidasas/efectos de los fármacos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Canales Aniónicos Dependientes del Voltaje/efectos de los fármacosRESUMEN
Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum-localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca2+ entry across the plasma membrane and the Ca2+ release-activated Ca2+ channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca2+ entry.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Células Endoteliales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Western Blotting , Células Cultivadas , Impedancia Eléctrica , Retículo Endoplásmico/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Técnica del Anticuerpo Fluorescente , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Proteínas de la Membrana/genética , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1 , Fosforilación , Interferencia de ARN , Transducción de Señal/genética , Transducción de Señal/fisiología , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6 , Trombina/farmacología , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca(2+)-activated K(+) channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K(+) channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca(2+)-activated K(+) channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/genética , Conductividad Eléctrica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioblastoma/cirugía , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/deficiencia , Datos de Secuencia Molecular , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genéticaRESUMEN
Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3H]aspartate efflux, and VRAC-mediated Cl- currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl- currents. The expression of candidate Cl- channels was confirmed by RT-PCR. The maxi Cl- channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl- channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl- currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl- currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.
Asunto(s)
Astrocitos/metabolismo , Canales de Cloruro/fisiología , Cloro/metabolismo , Fármacos actuantes sobre Aminoácidos Excitadores/administración & dosificación , Aminoácidos Excitadores/metabolismo , Activación del Canal Iónico/fisiología , Equilibrio Hidroelectrolítico/fisiología , Animales , Animales Recién Nacidos , Astrocitos/citología , Tamaño de la Célula , Células Cultivadas , Activación del Canal Iónico/efectos de los fármacos , Presión Osmótica , Ratas , Ratas Sprague-Dawley , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl- currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl- currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl- currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl- current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl- currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 micro M) was lower than that in control cells (31 micro M). However, the VSOR Cl- currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was markedly diminished by tyrphostin B46 but not significantly affected by tyrphostin AG1296. We thus conclude that the EGF receptor tyrosine kinase upregulates the activity of the VSOR Cl- channel, mainly by enhancing the volume sensitivity.
Asunto(s)
Canales de Cloruro/fisiología , Receptores ErbB/biosíntesis , Glándulas Mamarias Animales/citología , Regulación hacia Arriba/fisiología , Algoritmos , Animales , Línea Celular , Permeabilidad de la Membrana Celular/fisiología , Tamaño de la Célula/fisiología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Electrofisiología , Receptores ErbB/genética , Femenino , Glándulas Mamarias Animales/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Presión Osmótica , Técnicas de Placa-Clamp , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.