Vitamin D₃ analogs stimulate calcium currents in rat osteosarcoma cells

1α,25-Dihydroxyvitamin D₃ (1,25-D₃) rapidly (within seconds) shifts the threshold for activation of inward calcium currents to more negative and physiological potentials. To determine whether the opening of calcium channels in bone cells is mediated by the cytosolic 1,25-dihydroxyvitamin D₃ receptor (VDR), several natural metabolites 1,25-D₃, 25-hydroxyvitamin D₃, and 24R,25-dihydroxyvitamin D₃ and synthetic analogs 25-hydroxy-16,23E- diene D₃ (HO), 25-hydroxy-23-yne D₃ (Y), and 1α,25-dihydroxy-16-ene-23- yne-26,27-F6 D₃ (EO) were tested on dihydropyridine-sensitive inward barium currents. In order to probe the structural specificity at the 1 position of the steroid for stimulation of barium currents, we used several synthetic 1- (1'-hydroxyethyl) (NP, ON, NN, OP) and 1-(2'-hydroxyethyl)-25-hydroxyvitamin D₃ analogs (14w-1α and 14w-1β). Using the perforated patch-clamp technique, we found that the naturally occurring vitamin D₃ analogs gave nearly the same rank order potency for stimulation of barium currents as their affinity for VDR with 1,25-D₃ being the most potent analog. Using the synthetic analogs which have minimal affinity for VDR, we found that the compounds without 1-OH group but with additional double bonds in positions 16 and 23 or with a triple bond in position 23 retained high affinity for calcium channel activation. Furthermore, 1-hydroxyethyl-25-hydroxyvitamin D₃ R isomers at the 1' position had greater affinity than the S isomers at this position, and a β oriented 2'-hydroxyethyl group gave the homolog greater affinity than did the α-oriented 2'-hydroxyethyl group. The fact that these synthetic analogs cause rapid effects on calcium channels and show pharmacological specificity different from the binding to the cytosolic vitamin D₃ receptor suggests that calcium influx is mediated by a distinct signal transduction pathway. The high and physiological affinity of 1,25-D₃ (50 pM) suggests that it is a biological regulator of calcium channels.