ATP directly enhances calcium channels in the luminal membrane of the distal nephron

Calcium (Ca2+) transport by the distal tubule (DT) luminal membrane is regu lated by the parathyroid hormone (PTH) and calcitonin (CT) through the acti on of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytos olic surface of the membrane could influence the Ca2+ channels previously d etected in this membrane. We purified the luminal membranes of rabbit proxi mal (PT) and DT separately and measured Ca2+ uptake by these vesicles loade d with ATP or the carrier. The presence of 100 mu M ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca2+ uptake from 0.57 +/- 0.02 to 0. 71 +/- 0.02 pmol/mu g per 10 sec (P < 0.01) in the absence of Na+ and from 0.36 +/- 0.03 to 0.59 +/- 0.01 pmol/mu g per 10 sec (P < 0.01) in the prese nce of 100 mM Na+. This effect was dose dependent with an EC50 value of app roximately 40 mu M. ATP action involved the high-affinity component of Ca2 transport, decreasing the Km from 0.08 +/- 0.01 to 0.04 +/- 0.01 mM (P < 0 .02). Replacement of the nucleotide by the nonhydrolyzable ATP gamma s abol ished this action. Because ATP has been reported to be necessary for cytosk eleton integrity, we also investigated the effect of intravesicular cytocha lasin on Ca2+ transport. Inclusion of 20 mu M cytochalasin B decreased 0.5 mM Ca2+ uptake from 0.33 +/- 0.01 to 0.15 +/- 0.01 pmol/mu g per 10 sec (P < 0.01). However, when both 100 mu M ATP and 20 mu M cytochalasin were pres ent in the vesicles, the uptake was not different from that observed with A TP alone. Neither ATP nor cytochalasin had any influence on Ca2+ uptake by the PT luminal membrane. We conclude that the high-affinity Ca2+ channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial r ole in the integrity of the cytoskeleton which is also involved in the cont rol of Ca2+ channels within this membrane. (C) 1999 Wiley-Liss Inc.