Internal medicine

P-type calcium channel

Neuroscience

Receptor

Voltage-dependent calcium channel

Cinnarizine

Biology

Biophysics

Neurotransmission

Flunarizine

Calcium

T-type calcium channel

Biochemistry

Medicine

Calcium channel

Pharmacology

Cell Research

Dear Editor,Voltage-gated calcium (Ca v ) channels regulate a broad range of physiological processes, such as muscle contraction, synaptic signal transduction, and hormone secretion. 1Ca v 1.3 channels belong to the L-type (L for long-lasting current) Ca v subfamily (LTCC) and display diverse tissue distributions in brain, ear, heart, and endocrine organs. 2 Of note, Ca v 1.3 channels are predominantly expressed in cochlea inner hair cells, whose normal development and synaptic transmission are crucial for proper auditory function. 3herapeutic agents targeting Ca v 1.3 channels hold promises for Parkinson's disease, resistant hypertension, and hearing dysfunction. 4In addition to the widely prescribed dihydropyridine (DHP), phenylalkylamine (PAA), and benzothiazepine (BTZ) drugs, Ca v 1.3 channels are subject to regulation by compounds with the diphenylmethylpiperazine (DPP) core group, exemplified by cinnarizine and flunarizine (Supplementary information, Fig. S1). 5 DPP analogs, with their dual activities as antihistamines and Ca v channel blockers, are used to treat motion sickness, although the molecular details for their mode of actions (MOA) remain to be revealed. 6he rabbit Ca v 1.1 multimeric complex purified from endogenous skeletal muscle tissues has been employed as a prototype for structural analysis of LTCC antihypertensive and antiarrhythmic agents. 7Here, we present the high-resolution structures of human Ca v 1.3 complex, comprising the pore-forming α1 and auxiliary α2δ-1 and β3 subunits.Ligand-free and cinnarizine-bound human Ca v 1.3 structures were determined at 3.0 Å and 3.1 Å resolutions, respectively.To attain heterotrimeric Ca v 1.3 proteins, we transfected HEK293F cells with plasmids encoding full-length α1, α2δ-1 and β3 subunits at the mass ratio of 1.5:1.2:1.After tandem purification through affinity and size exclusion chromatography, peak fractions containing all three subunits were pooled for cryosample preparation (Supplementary information, Fig. S2a).The Ca v 1.3-drug samples were prepared by incubating concentrated proteins in the presence of 250 μM cinnarizine or 150 μM cp-PYT (compound 8 in 8 ), a pyrimidine-2,4,6-triones derivative that showed modest selective inhibition against Ca v 1.3.While cinnarizine is unambiguously resolved (Fig. 1a), there is no discernible density for cp-PYT in the electron microscopy (EM) map.cp-PYT was predicted to occupy the DHP-binding site, 7,9,10 which is on the interface of repeats III and IV in the pore domain (PD), known as the III-IV fenestration.As the entire PD is clearly resolved, the invisibility of cp-PYT is unlikely to result from potential flexibility.cp-PYT was shown to be a modest inhibitor of Ca v 1.3, with the IC 50 > 50 μM. 11,12It is possible that addition of 150 μM cp-PYT did not warrant a stable drug-channel complex that survived cryo-sample preparation.We will refer to the

Structural basis for pore blockade of human voltage-gated calcium channel Cav1.3 by motion sickness drug cinnarizine