Gain of channel function and modified gating properties in TRPM3 mutants causing intellectual disability and epilepsy

Abstract

33Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of disorders 34 characterized by epilepsy with comorbid intellectual disability. Recently, two de novo 35 heterozygous mutations in the gene encoding TRPM3, a calcium permeable ion channel, were 36 identified as the cause of DEE in eight probands, but the functional consequences of the 37 mutations remained elusive. Here we demonstrate that both mutations (V990M and P1090Q) 38 have distinct effects on TRPM3 gating, including increased basal activity, higher sensitivity to 39 stimulation by the endogenous neurosteroid pregnenolone sulphate (PS) and heat, and altered 40 response to ligand modulation. Most strikingly, the V990M mutation affected the gating of the 41 non-canonical pore of TRPM3, resulting in large inward cation currents via the voltage sensor 42 domain in response to PS stimulation. Taken together, these data indicate that the two DEE 43 mutations in TRPM3 result in a profound gain of channel function, which may lie at the basis 44 of epileptic activity and neurodevelopmental symptoms in the patients. 45 48 neurosteroid pregnenolone sulphate (PS) (2). TRPM3 is expressed in a large subset of mouse 49 and human somatosensory neurons, where it is involved in the detection of noxious heat and 50 the development of inflammatory pain (1, 3). Moreover, TRPM3 is expressed in several brain 51 areas, including the choroid plexus, cerebellum, cortex and the hippocampal formation (4-6), 52 but its functional role in these areas is unknown. Recently, two de novo substitutions in TRPM3 53 (V990M and P1090Q) were identified as the cause of intellectual disability and epilepsy in 54 eight probands with developmental and epileptic encephalopathy (DEE) (7). However, the 55 consequences of these mutations on TRPM3 function remained elusive. We here demonstrate 56 that both mutations lead to significant gain-of-channel function, including increased basal 57 activity and higher sensitivity to PS and heat. V990M exhibits further pronounced functional 58 alterations, including anomalous activation of the alternative current through the voltage-sensor 59 domain, reduced sensitivity to receptor-mediated inhibition and calcium-dependent 60 inactivation, and lower sensitivity to block by the anticonvulsant primidone.61 4 Results & Discussion 62When performing Fura-2-based calcium imaging on transiently transfected HEK293T cells, we 63 observed significantly higher intracellular Ca 2+ concentrations ([Ca 2+ ]i) in cells expressing the 64 two DEE mutants compared to wild type human TRPM3 (WT) ( Figure 1A,B), an effect that 65 was much more pronounced in the V990M mutant. Application of primidone or isosakuranetin, 66 both potent TRPM3 antagonists (8, 9), reduced [Ca 2+ ]i in cells expressing WT or mutant 67 TRPM3. In absolute terms, the antagonist-induced reduction in [Ca 2+ ]i was the largest for the 68 V990M mutant, yet [Ca 2+ ]i did not fully return to the level of cells expressing WT ( Figure 69 1A,C). These results suggest that the DEE...

Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants

“…The integration of cycles of computational and experimental analysis enabled us to provide mechanistic molecular insights into the different mechanisms by which two proximal candidate KCNC2 VUSs lead to DEE-like phenotypes. DEE has been previously linked to dysfunction of other ion channels 58,59 .…”

“…The integration of cycles of computational and experimental analysis enabled us to provide mechanistic molecular insights into the different mechanisms by which two proximal candidate KCNC2 VUSs lead to DEE-like phenotypes. DEE has been previously linked to dysfunction of other ion channels 58,59 . Moreover, Kv3 channel family members have been previously associated with neurological disorders such as ataxias, epilepsies, schizophrenia, and Alzheimer’s disease 60 .…”

TRPM3 Channels Play Roles in Heat Hypersensitivity and Spontaneous Pain after Nerve Injury

“…TRPM3 is expressed in the brain, and its overactive mutations cause intellectual disability and epilepsy in humans (Dyment et al, 2019;Van Hoeymissen et al, 2020;Zhao et al, 2020). Primidone crosses the blood-brain barrier (Nagaki et al, 1999), while specific data for isosakuranetin are not available, several very similar flavonoid molecules, such as liquiritigenin and naringenin (also TRPM3 inhibitors) have been shown to cross the blood-brain barrier (Youdim et al, 2003;Li et al, 2015a).…”

Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia

“…This nomenclature has been used by others (de Sainte Agathe et al, 2020; Gauthier et al, 2021) including Human Gene Mutation Database (Stenson et al, 2017) and OMIM (https://www.omim.org/). Others have reported this same variant as NM_001007471.2:c.2986G>A, NP_001007472.2:p.(Val990Met) (Van Hoeymissen et al, 2020; Zhao et al, 2020). In ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) the nomenclature used for this variant is NM_001366145.2:c.3004G>A; NP_001353074.1:p.(Val1002Met).…”

“…Subsequently, other individuals with a similar phenotype, though lacking epilepsy, were reported with the same recurrent variant (de Sainte Agathe et al, 2020; Gauthier et al, 2021). Although initial reports did not include any functional studies, subsequent in vitro experiments in heterologous expression systems demonstrated pathogenic gain‐of‐function through altered gating and conductance (Held et al, 2018; Van Hoeymissen et al, 2020; Zhao et al, 2020). The channels expressing the recurrent substitution exhibit increased basal activity, enhanced sensitivity to the TRPM3 agonist pregnenolone sulfate, increased thermal sensitivity, and increased permeability via a non‐canonical or “alternative” conductance pathway previously identified in TRPM3 (Van Hoeymissen et al, 2020; Vriens et al, 2014; Zhao et al, 2020).…”