Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity.

Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).