Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 1805K) PPT slides of all figures Supplemental Material Index Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Csanády, L. Articles by Adam-Vizi, V. Search for Related Content PubMed PubMed Citation Articles by Csanády, L. Articles by Adam-Vizi, V. Social Bookmarking                      What's this?

Department of Medical Biochemistry, Semmelweis University, and Neurochemical Group of the Hungarian Academy of Sciences, Budapest H-1444, Hungary

The online version of this article contains supplemental material

Regulation by cytosolic nucleotides of Ca²⁺- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (P_o) was suppressed by cytosolic nucleotides with apparent K_I values of 17, 9, and 2 µM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca²⁺] and voltage affected inhibition of P_o, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca²⁺ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3'-5' cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K_1/2 = 90 nM) and robustly stimulated P_o, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca²⁺ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates P_o with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned channels.

Key Words: microscopic reversibility • competitive binding • kinetic model • Trp channels • blood-brain-barrier

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				B. Nilius, J. Prenen, A. Janssens, T. Voets, and G. Droogmans Decavanadate modulates gating of TRPM4 cation channels J. Physiol., November 1, 2004; 560(3): 753 - 765. [Abstract] [Full Text] [PDF]

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents