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Hyperekplexia, an inherited neuronal disorder characterized by exaggerated startle responses with unexpected sensory stimuli, is caused by dysfunction of glycinergic inhibitory transmission. From analysis of newly identified human hyperekplexia mutations in the glycine receptor (GlyR) α1 subunit, we found that an alanine-to-proline missense mutation (A384P) resulted in substantially higher desensitization level and lower agonist sensitivity of homomeric α1 GlyRs when expressed in HEK cells. The incorporation of the β subunit fully reversed the reduction in agonist sensitivity and partially reversed the desensitization of α1 A384P The heteromeric α1 A384P β GlyRs showed enhanced desensitization but unchanged agonist-induced maximum responses, surface expression, main channel conductance, and voltage dependence compared with that of the wild-type α1β (α1 WT β) GlyRs. Coexpression of the R392H and A384P mutant α1 subunits, which mimic the expression of the compound heterozygous mutation in a hyperekplexia patient, resulted in channel properties similar to those with α1 A384P subunit expression alone. In comparison, another human hyperekplexia mutation α1 P250T , which was previously reported to enhance desensitization, caused a strong reduction in maximum currents in addition to the altered desensitization. These results were further confirmed by overexpression of α1 P250T or α1 A384P subunits in cultured neurons isolated from SD rats of either sex. Moreover, the IPSC-like responses of cells expressing α1 A384P β induced by repeated glycine pulses showed a stronger frequency-dependent reduction than those expressing α1 WT β. Together, our findings demonstrate that A384 is associated with the desensitization site of the α1 subunit and its proline mutation produced enhanced desensitization of GlyRs, which contributes to the pathogenesis of human hyperekplexia. SIGNIFICANCE STATEMENT Human startle disease is caused by impaired synaptic inhibition in the brainstem and spinal cord, which is due to either direct loss of GlyR channel function or reduced number of synaptic GlyRs. Considering that fast decay kinetics of GlyR-mediated inhibitory synaptic responses, the question was raised whether altered desensitization of GlyRs will cause dysfunction of glycine transmission and disease phenotypes. Here, we found that the α1 subunit mutation A384P, identified from startle disease patients, results in enhanced desensitization and leads to rapidly decreasing responses in the mutant GlyRs when they are activated repeatedly by the synaptic-like simulation. These observations suggest that the enhanced desensitization of postsynaptic GlyRs could be the primary pathogenic mechanism of human startle disease., (Copyright © 2018 the authors 0270-6474/18/382819-14$15.00/0.)

We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABA A ) receptor γ2 subunit gene GABRG2 in a patient with Dravet syndrome using targeted next-generation sequencing. The mutation was in the cytoplasmic portion of the transmembrane segment M2 of the γ2 subunit that faces the pore lumen. GABA A receptor α1 and β3 subunits were coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse cortical neurons. We measured currents using whole-cell and single-channel patch clamp techniques, surface and total expression levels using surface biotinylation and Western blotting, and potential structural perturbations in mutant GABA A receptors using structural modeling. The γ2(P302L) subunit mutation produced an ∼90% reduction of whole-cell current by increasing macroscopic desensitization and reducing GABA potency, which resulted in a profound reduction of GABA A receptor-mediated miniature IPSCs (mIPSCs). The conductance of the receptor channel was reduced to 24% of control conductance by shifting the relative contribution of the conductance states from high- to low-conductance levels with only slight changes in receptor surface expression. Structural modeling of the GABA A receptor in the closed, open, and desensitized states showed that the mutation was positioned to slow activation, enhance desensitization, and shift channels to a low-conductance state by reshaping the hour-glass-like pore cavity during transitions between closed, open, and desensitized states. Our study revealed a novel γ2 subunit missense mutation (P302L) that has a novel pathogenic mechanism to cause defects in the conductance and gating of GABA A receptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.

M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K(+) transport, and are regulated by numerous receptors via G(q/11)-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, and M current is known to be regulated by Ca(2+)/calmodulin (CaM) and PIP(2), we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca(2+)/CaM and PIP(2) on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M(1) receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to "sensitize" KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose-response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP(2) in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca(2+), to disrupt the interaction of YFP-tagged AKAP79(1-153) with CFP-tagged KCNQ2-5. Tonic depletion of PIP(2) by cotransfection of a PIP(2) phosphatase had no effect, and sudden depletion of PIP(2) did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP(2) depletion.

Heteromeric assembly of KCNQ2 and KCNQ3 subunits underlie the M-current (I(KM)), a slowly activating and noninactivating neuronal K(+) current. Mutations in KCNQ2 and KCNQ3 genes cause benign familial neonatal convulsions (BFNCs), a rare autosomal-dominant epilepsy of the newborn. In the present study, we describe the identification of a novel KCNQ2 heterozygous mutation (c587t) in a BFNC-affected family, leading to an alanine to valine substitution at amino acid position 196 located at the N-terminal end of the voltage-sensing S(4) domain. The consequences on KCNQ2 subunit function prompted by the A196V substitution, as well as by the A196V/L197P mutation previously described in another BFNC-affected family, were investigated by macroscopic and single-channel current measurements in CHO cells transiently transfected with wild-type and mutant subunits. When compared with KCNQ2 channels, homomeric KCNQ2 A196V or A196V/L197P channels showed a 20 mV rightward shift in their activation voltage dependence, with no concomitant change in maximal open probability or single-channel conductance. Furthermore, current activation kinetics of KCNQ2 A196V channels displayed an unusual dependence on the conditioning prepulse voltage, being markedly slower when preceded by prepulses to more depolarized potentials. Heteromeric channels formed by KCNQ2 A196V and KCNQ3 subunits displayed gating changes similar to those of KCNQ2 A196V homomeric channels. Collectively, these results reveal a novel role for noncharged residues in the N-terminal end of S(4) in controlling gating of I(KM) and suggest that gating changes caused by mutations at these residues may decrease I(KM) function, thus causing neuronal hyperexcitability, ultimately leading to neonatal convulsions.