RESUMO
Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.
Assuntos
Canais de Cálcio/metabolismo , Espermatozoides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Sinalização do Cálcio , AMP Cíclico/metabolismo , Feminino , Fertilização in vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transdução de Sinais , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidoresRESUMO
BACKGROUND: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (mCa2+) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. METHODS: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. RESULTS: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing mCa2+ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa2+ overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa2+ uptake impair mitochondrial energetics and contractile function. CONCLUSIONS: We detail a new regulatory mechanism to modulate mtCU function and mCa2+ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa2+ uptake and cellular physiology.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Sistemas CRISPR-Cas , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Técnicas de Inativação de Genes , Células HeLa , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Função Ventricular EsquerdaRESUMO
Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.