RESUMO
In this study we present 3 families with malignant hyperthermia (MH), all of Indian subcontinent descent. One individual from each of these families was fully sequenced for RYR1 and presented with the non-synonymous change c.11315G>A/p.R3772Q. When present in the homozygous state c.11315*A is associated with myopathic symptoms.
Assuntos
Hipertermia Maligna/genética , Doenças Musculares/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Adolescente , Adulto , Idoso , Povo Asiático , Consanguinidade , DNA/genética , DNA Complementar/biossíntese , DNA Complementar/genética , Família , Feminino , Genes Dominantes , Genes Recessivos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Doenças Musculares/congênito , Linhagem , Fenótipo , Espasmo/genética , Espasmo/patologiaRESUMO
Recombinant and native large conductance, Ca(2+)-activated K+ (BK) channels often demonstrate O2 sensitivity in cell-free membrane patches suggesting that a significant component of the O2-sensing machinery must be closely associated with the channel protein complex. Until recently, however, the identity of the O2 sensor itself had remained elusive. Employing functional proteomics we have defined the molecular nature of such an O2 sensor of BK channels. Using immunoprecipitation, 1D and 2D gel electrophoresis, and mass spectroscopy we identified the constitutive form of haem oxygenase, haem oxygenase 2 (HO-2), as a BK alpha-subunit protein partner. Functional measurement of hypoxic modulation of BK channel activity during manipulation of HO-2 enzyme substrates and reaction products, followed by protein knock-down of HO-2 using small interfering RNA, indicated that this enzyme is directly involved in hypoxic inhibition of BK channels. Furthermore, good correlation was observed between data obtained from recombinant BK channels and those from acutely isolated rat carotid body glomus cells, suggesting strongly that HO-2 also acts as an O2 sensor in native arterial chemoreceptors.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Oxigênio/metabolismo , Proteômica/métodos , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Oxigênio/fisiologiaAssuntos
Heme Oxigenase (Desciclizante)/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Oxigênio/metabolismo , Animais , Corpo Carotídeo/metabolismo , Hipóxia Celular , Linhagem Celular , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/genética , Humanos , RNA Interferente Pequeno/genética , Ratos , TransfecçãoRESUMO
Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.
Assuntos
Monóxido de Carbono/metabolismo , Corpo Carotídeo/citologia , Corpo Carotídeo/fisiologia , Heme Oxigenase (Desciclizante)/metabolismo , Oxigênio/fisiologia , Animais , Hipóxia Celular , Linhagem Celular , Heme/metabolismo , Heme Oxigenase (Desciclizante)/genética , Humanos , Imunoprecipitação , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana , NADP/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , TransfecçãoRESUMO
Large conductance, Ca(2+)-sensitive potassium (BK) channels are critical components of the O(2) signalling cascade in a number of cells, including the carotid body and central neurones. Although the nature of the BK channel O(2) sensor is still unknown, evidence suggests redox modulators might form part of the O(2) sensing channel complex. By metabolising glutathione, gamma-glutamyl transpeptidase (gammaGT) could act as such an O(2) sensor. Western blotting and immunocytochemistry revealed high gammaGT expression in HEK293 cells expressing the alpha- and beta-subunits of human recombinant BK and gammaGT co-immunoprecipitated with BKalpha. Acivicin blockade of gammaGT reversibly inhibited BK channels, suggesting that this BKalpha protein partner contributes to tonic channel activity. However, knock-out of gammaGT using siRNA had no effect on hypoxic BK channel inhibition. Together, these data indicate that gammaGT is a BKalpha protein partner, that its activity regulates BK channels but that it is not the BK O(2) sensor.