Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide

Abstract

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca²⁺-sensitive regulators, each with three Ca²⁺-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca²⁺/CaM (calmodulin)-dependent protein kinase] and its PpCaMK (Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca²⁺-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca²⁺-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC₅₀ values of 400 μM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca²⁺-dependent manner and antagonized the regulatory function of the domain with IC₅₀ values of 55 and 4 μM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K_ATP channels with similar potency, glibenclamide had no antagonizing effect on the Ca²⁺-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca²⁺-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists.