RESUMO
An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and ß3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.
Assuntos
Processamento Alternativo , Canais de Cálcio Tipo L/química , Biofísica/métodos , Encéfalo/metabolismo , Cálcio/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Linhagem Celular , Clonagem Molecular , Células HEK293 , Humanos , Íons , Neurônios/metabolismo , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , RNA/metabolismo , Transdução de SinaisRESUMO
Auxiliary beta subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca(2+) channels in heterologous cells. In brain, all four beta isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of beta subunits and their role in membrane expression of Ca(V)1.2 alpha(1) subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous beta subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged beta subunits demonstrated that all beta subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca(V)1.2, thus indicating a great promiscuity in alpha(1)-beta interactions. In contrast, restricted axonal targeting of beta(1) and weak colocalization of beta(4b) with Ca(V)1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca(V)1.2 was strongly enhanced by all beta subunits in an isoform-specific manner. Conversely, mutating the alpha-interaction domain of Ca(V)1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a beta subunit with the alpha-interaction domain is absolutely essential for membrane expression of alpha(1) subunits, as well as for the subcellular localization of beta subunits, which by themselves possess little or no targeting properties.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Hipocampo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Subunidades Proteicas/metabolismo , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo L/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína/fisiologia , Subunidades Proteicas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca(v)1.3 voltage-gated L-type Ca(2+) channels. Their voltage- and Ca(2+)-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2alpha (RIM2alpha) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca(v)1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the beta-subunit of the channel complex and RIM-induced slowing of both Ca(2+)- and voltage-dependent inactivation of Ca(v)1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca(v)1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca(v)1.3 gating kinetics in immature IHCs.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células Ciliadas Auditivas Internas/fisiologia , Ativação do Canal Iônico/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Potenciais de Ação/fisiologia , Processamento Alternativo , Animais , Canais de Cálcio Tipo L/genética , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Células Ciliadas Auditivas Internas/citologia , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Ca(v)1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and alpha1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Ca(v)1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human alpha1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Ca(v)1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Sítios de Ligação/genética , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Calmodulina/farmacologia , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Ativação do Canal Iônico/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Microscopia Confocal/métodos , Mutação de Sentido Incorreto/genética , Técnicas de Patch-Clamp , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Ca(v)1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Ca(v)1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d(-/-) mice.
Assuntos
Bradicardia/fisiopatologia , Canais de Cálcio Tipo L/fisiologia , Canalopatias/fisiopatologia , Surdez/fisiopatologia , Adolescente , Adulto , Bradicardia/genética , Canais de Cálcio Tipo L/genética , Canalopatias/genética , Surdez/congênito , Surdez/genética , Feminino , Células HEK293 , Células Ciliadas Auditivas Internas/fisiologia , Haplótipos , Humanos , Masculino , Mutação , Linhagem , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Nó Sinoatrial/fisiologia , Síndrome , Transfecção/métodosRESUMO
Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta(4) subunit gene cause ataxia and epilepsy. However, the specific function of beta(4) in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta(4) subunit in the nuclei of cerebellar granule and Purkinje cells. beta(4b) was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta(4b) nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta(4b) into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Músculos/metabolismo , Neurônios/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ratos , Ratos WistarRESUMO
Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.