THERAPEUTIC POTENTIAL OF MPI INHIBITION TO REVERT PATHOGENIC CA<SUB>V</SUB>2.1 GAIN-OF-FUNCTION IN <EM>CACNA1A</EM>-RELATED NEUROLOGICAL DISORDERS

The CACNA1A gene encodes the α_1A subunit of the Ca_V2.1 (P/Q-type) voltage-gated calcium channel. Gain-of-function mutations in CACNA1A cause neurological disorders such as Familial Hemiplegic Migraine type 1 (FHM1) and Congenital Ataxia (CA) by increasing calcium influx and favouring neuronal excitability, which in turn promote cortical spreading depression (migraine pathophysiology) and Purkinje cell hyperexcitability (ataxia). Hypoglycosylation of Ca_V2.1, observed in Phosphomannomutase 2 deficiency (PMM2-CDG), mimics this effect. Since Phosphomannose Isomerase (MPI) competes with PMM2 for mannose-6-phosphate, we hypothesized that MPI inhibition combined with mannose supplementation could enhance N-glycosylation and counteract channel gain-of-function.
Using HEK293 cells expressing wild-type or mutant (Y1245C, Δ1502) Ca_V2.1, we tested the effect of MPI inhibitor MLS0315771 with mannose on N-glycosylation (Western Blot) and channel function (whole-cell patch-clamp electrophysiology). Treatment increased N-glycosylation of the α₂δ subunit (p<0.01, n=4). The Δ1502 mutation caused a ~26 mV leftward shift in activation voltage (p<0.01, n=3-13) and slowed inactivation (p<0.05, n=5-11), both hallmarks of pathological gain-of-function. MPI inhibition with mannose partially normalized activation (by 6.5 mV, p<0.05, n=4) and inactivation kinetics (p<0.05, n=4-5). The Y1245C mutation reduced activation voltage (by ~6 mV, p<0.05, n=7-13), but treatment did not alter this effect. PMM2 silencing by siRNA decreased α₂δ subunit N-glycosylation and membrane trafficking (p<0.05, n=3) and slowed inactivation (p<0.05, n=16-18), mimicking gain-of-function, which was reversed by MPI inhibition plus mannose (p<0.05, n=9-16).
These findings show that enhancing N-glycosylation via MPI inhibition mitigates pathogenicgain-of-function in specific CACNA1A mutations and PMM2 deficiency, proposing a targeted therapeutic approach for Ca_V2.1-related channelopathies.
Funding: MCIN/AEI/10.13039/501100011033; FEDER (PID2022-136546OB-I00).