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ATP directly enhances calcium channels in the luminal membrane of the distal nephron
Author(s) -
Brunette M.G.,
Mailloux J.,
Hilal G.
Publication year - 1999
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199912)181:3<416::aid-jcp5>3.0.co;2-x
Subject(s) - cytochalasin b , chemistry , vesicle , calcium , biophysics , cytosol , membrane , apical membrane , membrane transport , cytochalasin , biochemistry , cytoskeleton , biology , enzyme , in vitro , cell , organic chemistry
Calcium (Ca 2+ ) transport by the distal tubule (DT) luminal membrane is regulated by the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytosolic surface of the membrane could influence the Ca 2+ channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca 2+ uptake by these vesicles loaded with ATP or the carrier. The presence of 100 μM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca 2+ uptake from 0.57 ± 0.02 to 0.71 ± 0.02 pmol/μg per 10 sec ( P < 0.01) in the absence of Na + and from 0.36 ± 0.03 to 0.59 ± 0.01 pmol/μg per 10 sec ( P < 0.01) in the presence of 100 mM Na + . This effect was dose dependent with an EC 50 value of approximately 40 μM. ATP action involved the high‐affinity component of Ca 2+ 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 ATPγs abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, we also investigated the effect of intravesicular cytochalasin on Ca 2+ transport. Inclusion of 20 μM cytochalasin B decreased 0.5 mM Ca 2+ uptake from 0.33 ± 0.01 to 0.15 ± 0.01 pmol/μg per 10 sec ( P < 0.01). However, when both 100 μM ATP and 20 μM cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca 2+ uptake by the PT luminal membrane. We conclude that the high‐affinity Ca 2+ channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca 2+ channels within this membrane. J. Cell. Physiol. 181:416–423, 1999. © 1999 Wiley‐Liss, Inc.