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