1,4-Dihydropyridine binding sites in moss plasma membranes: Properties of receptors for a calcium channel antagonist

University of Arizona,. An increase in cytoplasmic calcium is an early event in hormone (cytokinin)-induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport studies have implicated 1,4-dihydropyridine-sensitive calcium channels in this increase in cellular calcium. To understand the molecular nature of the dihydropyridine-sensitive calcium channel, we have established conditions for the binding of the arylazide 1,4-dihydropyridine, [³H]azidopine, to its receptor in moss plasma membranes. [³H]Azidopine bound specifically in a saturable and reversible manner. The K_D for [³H]azidopine binding was 5.2 nM and the B_max was 35.6 pmol/mg of protein. Association and dissociation of the receptor and [³H]azidopine were temperature-dependent, and association varied as a function of pH. Binding was inhibited by dihydropyridine, phenylalkylamine, and benzothiazepine calcium channel blockers, bepridil, lanthanum, and N-ethylmaleimide. [³H]Azidopine binding was stimulated by cations including calcium, strontium, manganese, and barium. [³H]Azidopine binding was also stimulated by cytokinin with a K_m value for kinetin of 0.13 nM. These studies utilize a simple plant system to provide a biochemical framework for understanding calcium regulation during development and have implications for understanding mechanisms of signal transduction in plants.