Molecular pharmacology of the amiloride analog 3-amino-6-chloro-5-[(4- chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)-amino]iminomethyl] -pyrazinecarboxamide (CB-DMB) as a pan inhibitor of the Na+-Ca 2+ exchanger isoforms NCX1, NCX2, and NCX3 in stably transfected cells

With the help of single-cell microflorimetry, ⁴⁵Ca²⁺ radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]- N-[[(2,4-dimethylbenzyl)amino]-iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na⁺/Ca²⁺ exchanger (NCX) and on several other membrane currents including voltage- and pHsensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC₅₀ values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the α₂ repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca²⁺ channels, tetrodotoxin-sensitive Na⁺ channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CBDMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.