RESUMEN
Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in CaV2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in CaV2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat CaV2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an â¼25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of CaV2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type CaV2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in CaV2.1.
Asunto(s)
Canales de Calcio Tipo N/genética , Activación del Canal Iónico/genética , Mutación/genética , Trastornos del Neurodesarrollo/genética , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Mutación/fisiología , Trastornos del Neurodesarrollo/fisiopatología , Unión Neuromuscular/genética , Unión Neuromuscular/fisiopatología , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Terminales Presinápticos/fisiología , Conejos , Ratas , Sinapsis/genética , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an "orthograde" signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a "retrograde" signal from RyR1 increases the amplitude of the Ca2+ current produced by CaV1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with CaV1.1 by producing an N-terminal construct (RyR11:4300) that lacks the C-terminal membrane domain. In CaV1.1-null (dysgenic) myotubes, RyR11:4300 is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR-PM junctions containing endogenous CaV1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR11:4300 is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR11:4300 stably associates with PM-SR junctions. Strikingly, expression of RyR11:4300 in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through CaV1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that â¼25% of RyR11:4300 in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR11:4300 is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DHPR tetrads is substantially increased by transfecting RyR-null myotubes with RyR11:4300 In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR-PM junctions in myotubes only if CaV1.1 is also present and is fully functional in retrograde signaling to CaV1.1.