Inhibition of Ca _V 1.4 channels by Ca _V 3 channel antagonists ML218 and Z944

Among the three classes of voltage-gated Ca ²⁺ channels (Ca _v 1, Ca _v 2, Ca _v 3), Ca _v 3 T-type channels are drug targets for disorders including epilepsy and pain. Antagonists such as Z944 and ML218 are highly selective for Ca _v 3 compared to the Ca _v 1.2 L-type channel but whether they have additional activity on other Ca _v 1 subtypes is unknown. Here, we investigated the effects of Z944 and ML218 on the Ca _v 1.4 channel which regulates neurotransmitter release from retinal photoreceptors. In HEK293T cells transfected with Ca _v 1.4 and the auxiliary β _2×13 and α ₂ δ-4 subunits, Z944 and ML218 inhibited Ca ²⁺ currents with IC ₅₀ values of ∼30 µM 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 to mutation of a conserved threonine (T1007) that is required for DHP sensitivity of Ca _v 1 channels, mutation of M1004 had a 10-fold greater impact in diminishing the potency of ML218. Ca _v 1.2 was significantly less sensitive to ML218 inhibition (IC ₅₀ ∼ 37 µM) than Ca _v 1.4, which could not be attributed to a valine in place of M1004 in Ca _v 1.2. We conclude that ML218 and Z944 are dual Ca _v 1/Ca _v 3 modulators of Ca _V 1.4 and should be used with caution when dissecting the contributions of Ca _V 3 channels in tissues where Ca _v 1.4 is expressed.