A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

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 ∼25-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.

Congenital myasthenic syndrome: a brief review.

Congenital myasthenic syndromes comprise heterogeneous genetic diseases characterized by compromised neuromuscular transmission. Congenital myasthenic syndromes are classified as presynaptic, synaptic, or postsynaptic, depending on the primary defect's location within the neuromuscular junction. Presynaptic forms are the rarest, affecting an estimated 7-8% of patients; synaptic forms account for approximately 14-15% of patients; and the remaining 75-80% are attributable to postsynaptic defects. Clinical manifestations vary by congenital myasthenic syndrome subtype. Electrophysiologic, morphologic, and molecular descriptions of various forms of congenital myasthenic syndromes have led to an enhanced understanding of clinical manifestations and disease pathophysiology. Although congenital myasthenic syndromes are indicated by clinical manifestations, family history, electrophysiologic studies, and responses to acetylcholinesterase inhibitors, overlap in some presentations occurs. Therefore, genetic testing may be necessary to identify specific mutations in CHAT, COLQ, LAMB2, CHRNA, CHRNB, CHRND, CHRNE, CHRNG, RAPSN, DOK7, MUSK, AGRN, SCN4A, GFPT1, or PLEC1 genes. The identification of congenital myasthenic syndromes subtypes will prove important in the treatment of these patients. Different drugs may be beneficial, or should be avoided because they are ineffective or worsen some forms of congenital myasthenic syndromes. We explore the classification, clinical manifestations, electrophysiologic features, genetics, and treatment responses of each congenital myasthenic syndrome subtype.

Morphological aspects of neuromuscular junctions and gene expression of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle of rats with heart failure.

HF is syndrome initiated by a reduction in cardiac function and it is characterized by the activation of compensatory mechanisms. Muscular fatigue and dyspnoea are the more common symptoms in HF; these may be due in part to specific skeletal muscle myopathy characterized by reduced oxidative capacity, a shift from slow fatigue resistant type I to fast less fatigue resistant type II fibers and downregulation of myogenic regulatory factors (MRFs) gene expression that can regulate gene expression of nicotinic acetylcholine receptors (nAChRs). In chronic heart failure, skeletal muscle phenotypic changes could influence the maintenance of the neuromuscular junction morphology and nAChRs gene expression during this syndrome. Two groups of rats were studied: control (CT) and Heart Failure (HF), induced by a single intraperitoneal injection of monocrotaline (MCT). At the end of the experiment, HF was evaluated by clinical signs and animals were sacrificed. Soleus (SOL) muscles were removed and processed for morphological, morphometric and molecular NMJ analyses. Our major finding was an up-regulation in the gene expression of the alpha1 and epsilon subunits of nAChR and a spot pattern of nAChR in SOL skeletal muscle in this acute monocrotaline induced HF. Our results suggest a remodeling of nAChR alpha1 and epsilon subunit during heart failure and may provide valuable information for understanding the skeletal muscle myopathy that occurs during this syndrome.

Structural and functional organization of synaptic acetylcholinesterase.

The expression of the synaptic asymmetric form of the enzyme acetylcholinesterase (AChE) depends of two different genes: the gene that encodes for the catalytic subunit and the gene that encodes for the collagenic tail, ColQ. Asymmetric AChE is specifically localized to the basal lamina at the neuromuscular junction (NMJ). This highly organized distribution pattern suggests the existence of one or more specific binding sites in ColQ required for its anchorage to the synaptic basal lamina. Recent evidence support this notion: first, the presence of two heparin-binding domains in ColQ that interact with heparan sulfate proteoglycans (HSPGs) at the synaptic basal lamina; and second, a knockout mouse for perlecan, a HSPG concentrated in nerve-muscle contact, in which absence of asymmetric AChE at the NMJ is observed. The physiological importance of collagen-tailed AChE form in skeletal muscle has been illustrated by the identification of several mutations in the ColQ gene. These mutations determine end-plate acetylcholinesterase deficiency and induce one type of synaptic functional disorders observed in Congenital Myasthenic Syndromes (CMSs).

Ca2+ channels and synaptic transmission at the adult, neonatal, and P/Q-type deficient neuromuscular junction.

Different types of voltage-activated Ca(2+) channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q-type, is the main channel involved in nerve-evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton-Lambert Syndrome. At adult neuromuscular junctions, L- and N-type Ca(2+) channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve-evoked synaptic transmission depends jointly on both N- and P/Q-type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q-type Ca(2+) channel knockout mice is also dependent on two different types of channels, N- and R-type. At both neonatal and P/Q knockout junctions, the K(+)-evoked increase in miniature endplate potential frequency was not affected by N-type channel blockers, but strongly reduced by both P/Q- and R-type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q- or R-type Ca(2+) channels located closer to the release site than N-type Ca(2+) channels. Thus, Ca(2+) channels may be recruited to mediate neurotransmitter release where P/Q-type channels seem to be the most suited type of Ca(2+) channel to mediate exocytosis at neuromuscular junctions.