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Investigating the Mechanism by Which Gain-of-function Mutations to the ?1 Glycine Receptor Cause Hyperekplexia.

ABSTRACT: Hyperekplexia is a rare human neuromotor disorder caused by mutations that impair the efficacy of glycinergic inhibitory neurotransmission. Loss-of-function mutations in the GLRA1 or GLRB genes, which encode the ?1 and ? glycine receptor (GlyR) subunits, are the major cause. Paradoxically, gain-of-function GLRA1 mutations also cause hyperekplexia, although the mechanism is unknown. Here we identify two new gain-of-function mutations (I43F and W170S) and characterize these along with known gain-of-function mutations (Q226E, V280M, and R414H) to identify how they cause hyperekplexia. Using artificial synapses, we show that all mutations prolong the decay of inhibitory postsynaptic currents (IPSCs) and induce spontaneous GlyR activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as the depleted chloride gradient should also lead to pain sensitization and to a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients with GLRA1 hyperekplexia mutations. We also rule out small increases in IPSC decay times (as caused by W170S and R414H) as a possible mechanism given that the clinically important drug, tropisetron, significantly increases glycinergic IPSC decay times without causing motor side effects. A recent study on cultured spinal neurons concluded that an elevated intracellular chloride concentration late during development ablates ?1? glycinergic synapses but spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is primarily responsible for the hyperekplexia phenotype.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC4946944 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia.

Zhang Yan Y Bode Anna A Nguyen Bindi B Keramidas Angelo A Lynch Joseph W JW

The Journal of biological chemistry 20160518 29

Hyperekplexia is a rare human neuromotor disorder caused by mutations that impair the efficacy of glycinergic inhibitory neurotransmission. Loss-of-function mutations in the GLRA1 or GLRB genes, which encode the α1 and β glycine receptor (GlyR) subunits, are the major cause. Paradoxically, gain-of-function GLRA1 mutations also cause hyperekplexia, although the mechanism is unknown. Here we identify two new gain-of-function mutations (I43F and W170S) and characterize these along with known gain-o ...[more]

PMID: 27226610

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Project description:T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.

Dataset Information

Investigating the Mechanism by Which Gain-of-function Mutations to the ?1 Glycine Receptor Cause Hyperekplexia.

Publications

Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia.

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