RESUMO
Ca(V)2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca(V)2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca(V)2 channels. Here we show that Ca(V)2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca(V)2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca(V)2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a--not e37b--is selected during alternative splicing of Ca(V)2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca(V)2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca(V)2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação/fisiologia , Processamento Alternativo , Animais , Canais de Cálcio Tipo N/genética , Gânglios Espinais/metabolismo , Camundongos , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances µ-opioid receptor-mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Canais de Cálcio Tipo N/genética , Morfina/farmacologia , Medula Espinal/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Interações Medicamentosas , Estimulação Elétrica/métodos , Embrião de Mamíferos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Éxons/genética , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/genética , Hiperalgesia/patologia , Lectinas/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Medula Espinal/citologia , Medula Espinal/fisiologia , ômega-Conotoxina GVIA/farmacologiaRESUMO
Carbon nanotubes (CNTs) are used with increasing frequency in neuroengineering applications. CNT scaffolds are used to transmit electrical stimulation to cultured neurons and to control outgrowth and branching patterns of neurites. CNTs have been reported to disrupt normal neuronal function including alterations in endocytotic capability and inhibition of ion channels. Calcium ion channels regulate numerous neuronal and cellular functions including endo and exocytosis, neurite outgrowth, and gene expression. Strong CNT interactions with neuronal calcium ion channels would have profound biological implications. Here we show that physiological solutions containing CNTs inhibit neuronal voltage-gated calcium ion channels in a dose-dependent and CNT sample-dependent manner with IC50 as low as 1.2 microg/ml. Importantly, we demonstrate that the inhibitory activity does not involve tubular graphene as previously reported, but rather very low concentrations of soluble yttrium released from the nanotube growth catalyst. Cationic yttrium potently inhibits calcium ion channel function with an inhibitory efficacy, IC50, of 0.07 ppm w/w. Because of this potency, unpurified and even some reportedly "purified" CNT samples contain sufficient bioavailable yttrium to inhibit channel function. Our results have important implications for emerging nano-neurotechnology and highlight the critical role that trace components can play in the biological response to complex nanomaterials.