STATE-DEPENDENT CA<SUB>V</SUB>2.1 INHIBITION CORRECTS NEURONAL CA²⁺ OVERLOAD IN CULTURED HEMIPLEGIC MIGRAINE MOUSE NEURONS AND MITIGATES HYPERTRANSMISSION IN LIVE <EM>C. </EM><EM>ELEGANS</EM> CARRYING A GAIN-OF-FUNCTION <EM>CACNA1A</EM> MUTATION

Hemiplegic migraine (HM), a severe migraine subtype with transient hemiparesis, is primarily caused by gain-of-function mutations in CACNA1A, encoding the Ca_V2.1 channel. Existing Ca_V2.1 inhibitors show poor bioavailability and limited brain penetration. We aimed to develop state-dependent Ca_V2.1 inhibitors as new therapeutic approach for HM.
Eight small molecules derived from a known Ca_V inhibitor were screened by automated patch-clamp in CHO cells expressing human Ca_V2.1, Ca_V1.2, or Ca_V2.2. Selected compounds were further tested in HEK293 cells expressing wild-type or HM-associated Y1245C Ca_V2.1. Cultured cortical neurons from wild-type and Ca_V2.1-R192Q knock-in mouse embryos were analyzed by calcium imaging after high-potassium stimulation. C. elegans strains carrying the human-equivalent Ca_V2.1-D1640N gain-of-function mutation or wild-type were assessed for aldicarb sensitivity to evaluate synaptic neurotransmission in vivo. Experiments complied with EU Directive 2010/63/EU.
Compounds UOS-00021171 and UOS-00021174, selectively inhibited Ca_V2.1 over other Ca_V channels and reduced mutant over wild-type Ca_V2.1 currents. At +5 mV, UOS-00021171 reduced maximal Ca²⁺ current by 31±8% (Y1245C) versus 5±7% (wild-type; n=6-8), and UOS-00021174 by 49±5% versus 24±11% (n=6-7, p<0.05).
UOS-00021174 normalized abnormally elevated Ca²⁺ responses in R192Q neurons, and reduced aldicarb hypersensitivity in live D1640N C. elegans (p<0.05) (indicating correction of excessive cholinergic transmission at nematode neuromuscular junctions), with minimal impact on wild-type neurons for in vitro and in vivo studies.
These findings identify UOS-00021174 as a promising HM therapeutic candidate. State-dependent Ca_V2.1 inhibition corrects pathological neuronal hyperactivity while preserving physiological function, suggesting fewer side effects than current treatments.
Funding: MCIN/AEI/10.13039/501100011033, FEDER (PID2022-136546OB-I00); Migraine Research Foundation.