RESUMEN
The fluorescent dye fura-2 AM was employed to record activation of Ca2+ entry in response to a decrease in Ca2+ concentration in the endoplasmic reticulum. Using whole-cell voltage clamp technique, we revealed Ca2+ currents with an amplitude of 0.46±0.13 pA/pF that passed through selective channels with current-voltage characteristics similar to those of classical store-operated CRAC channels. These currents were sensitive to 2-APB (50 µM), an inhibitor of store-operated channels. The data suggest that store-operated calcium entry is a characteristic feature of mature ventricular cardiomyocytes. Pathological alterations in store-operated Ca2+ entry can be implicated in the development of heart diseases.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Transporte Iónico/fisiología , Miocitos Cardíacos/fisiología , Animales , Células Cultivadas , Fura-2/análogos & derivados , Fura-2/farmacología , Ventrículos Cardíacos/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Función VentricularRESUMEN
Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells. Monomeric Homer 1a proteins increase activity of Imin channels, but did not modulate their electrophysiological properties. Recombinant Homer 1c protein did not block the induced calcium currents.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas de Andamiaje Homer/fisiología , Potenciales de Acción/efectos de los fármacos , Agonistas de los Canales de Calcio/metabolismo , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Citoplasma/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Proteínas de Andamiaje Homer/farmacología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Técnicas de Placa-Clamp , Multimerización de Proteína/fisiología , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.
Asunto(s)
Canales de Calcio/genética , Calcio/metabolismo , Retículo Endoplásmico/fisiología , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1/genética , Canales de Calcio/metabolismo , Señalización del Calcio , Células Clonales , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Transporte Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Proteínas de Neoplasias/deficiencia , Técnicas de Placa-Clamp , Molécula de Interacción Estromal 1/deficiencia , Tapsigargina/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Alzheimer's disease is the most common neurodegenerative disorder characterized by progressive memory and cognitive abilities loss. The etiology of Alzheimer's disease is poorly understood. In this regard, there is no effective treatment for the disease. Various hypotheses to explain the nature of the pathology of Alzheimer's disease led to the development of appropriate therapeutics. Despite of decades of research and clinical trials available therapeutics, at best, can only slow down the progression of the disease, but cannot cure it. This review dedicated to the one of modern hypotheses of Alzheimer's disease pathogenesis implied the impairment of calcium homeostasis as a key event for the development of neurodegenerative processes.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Canales de Calcio/genética , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Cognición , Marcadores Genéticos , Hipocampo/metabolismo , Hipocampo/patología , Homeostasis , Humanos , Memoria , Presenilina-1/genética , Proteínas tau/genética , Proteínas tau/metabolismoAsunto(s)
Señalización del Calcio , Enfermedad de Huntington/metabolismo , Modelos Neurológicos , Línea Celular , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Placa-Clamp , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Activation of phospholipase C-coupled receptors leads to the release of Ca2+ from Ca2+ stores, and subsequent activation of store-operated cation (SOC) channels, promoting sustained Ca2+ influx. The most studied SOC channels are CRAC ("calcium-release activated calcium") channels exhibiting a very high selectivity for Ca2+. However, there are many SOC channels permeable for Ca2+ but having a lower selectivity. And while Ca2+ influx is important for many biological processes, little is known about the types of SOC channels and mechanisms of SOC channel activation. Previously, we described store-operated Imin channels in A431 cells. Here, by whole-cell recordings, we demonstrated that the store depletion activates two types of current in A431 cells--highly selective for divalent cations (presumably, ICRAC), and moderately selective (ISOC supported by Imin channels). These currents can be registered separately and have different developing time and amplitude. Coexisting of two different types of SOC channels in A431 cells seems to facilitate the control of intracellular Ca(2+)-dependent processes.
Asunto(s)
Canales de Calcio/metabolismo , Línea Celular Tumoral/metabolismo , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Cationes , Citoplasma/metabolismo , Conductividad Eléctrica , Humanos , Estimulación QuímicaRESUMEN
Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.
Asunto(s)
Actinas/fisiología , Canales de Calcio/metabolismo , Calcio/metabolismo , Citoesqueleto/fisiología , Actinas/efectos de los fármacos , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Señalización del Calcio , Carcinoma de Células Escamosas/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Conductividad Eléctrica , Espacio Extracelular/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/farmacología , Activación del Canal Iónico , Toxinas Marinas , Oxazoles/farmacología , Técnicas de Placa-Clamp , Tapsigargina/farmacología , Células Tumorales CultivadasRESUMEN
I(min) is a plasma membrane-located, Ca(2+)-selective channel that is activated by store depletion and regulated by inositol 1,4, 5-trisphosphate (IP(3)). In the present work we examined the coupling between I(min) and IP(3) receptors in excised plasma membrane patches from A431 cells. I(min) was recorded in cell-attached mode and the patches were excised into medium containing IP(3). In about 50% of experiments excision caused the loss of activation of I(min) by IP(3.) In the remaining patches activation of I(min) by IP(3) was lost upon extensive washes of the patch surface. The ability of IP(3) to activate I(min) was restored by treating the patches with rat cerebellar microsomes reach in IP(3) receptors but not by control forebrain microsomes. The re-activated I(min) had the same kinetic properties as I(min) when it is activated by Ca(2+)-mobilizing agonists in intact cells and by IP(3) in excised plasma membrane patches and it was inhibited by the I(crac) inhibitor SKF95365. We propose that I(min) is a form of I(crac) and is gated by IP(3) receptors.