Inhibition of Ca_V1.4 channels by Ca_V3 channel antagonists ML218 and Z944

Among the three classes of voltage-gated Ca²⁺ channels (Ca_V1, Ca_V2, and Ca_V3), Ca_V3 T-type channels are drug targets for disorders, including epilepsy and pain. Antagonists such as Z944 and ML218 are highly selective for Ca_V3 compared with the Ca_V1.2 L-type channel, but whether they have additional activity on other Ca_V1 subtypes is unknown. Here, we investigated the effects of Z944 and ML218 on the Ca_V1.4 channel, which regulates neurotransmitter release from retinal photoreceptors. In HEK293T cells transfected with Ca_V1.4 and the auxiliary β_2x13 and α₂δ-4 subunits, Z944 and ML218 inhibited Ca²⁺ currents with IC₅₀ values of ∼30 and 2 µM, respectively. Structure-based modeling combined with functional studies revealed the importance of a cluster of methionine residues, particularly M1004, within the DHP-binding site for the effects of ML218. Compared with mutation of a conserved threonine (T1007) that is required for DHP sensitivity of Ca_V1 channels, mutation of M1004 had a 10-fold greater impact in diminishing the potency of ML218. Ca_V1.2 was significantly less sensitive to ML218 inhibition (IC₅₀ ∼37 µM) than Ca_V1.4, which could not be attributed to a valine in place of M1004 in Ca_V1.2. We conclude that ML218 and Z944 are dual Ca_V1/Ca_V3 modulators of Ca_V1.4 and should be used with caution when dissecting the contributions of Ca_V3 channels in tissues where Ca_V1.4 is expressed.