1,4-DIHYDROPYRIDINE BINDING-SITES IN MOSS PLASMA-MEMBRANES - PROPERTIES OF RECEPTORS FOR A CALCIUM-CHANNEL ANTAGONIST

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, [H-3]azidopine, to its receptor in moss plasma membranes, [H-3]Azidopine bound specifically in a saturable and reversible manner, The K-D for [H-3]azidopine binding was 5.2 nM and the B-max was 35.6 pmol/mg of protein, Association and dissociation of the receptor and [H-3]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. [H-3]Azidopine binding was stimulated by cations including calcium, strontium, manganese, and barium, [H-3]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.