TRINITROPHENYL-ATP BLOCKS COLONIC CL- CHANNELS IN PLANAR PHOSPHOLIPID-BILAYERS

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulat ion and transepithelial transport. Several recent reports indicate tha t rectifying Cl- channels are blocked after addition of ATP to the ext racellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D . M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137- 159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6-trinitrocyclohexa dienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high aff inity for certain nucleotide binding sites. Here we report that TNP-AT P blocked colonic Cl- channels when added to either bath and that bloc kade was consistent with the closed-open-blocked kinetic model. The TN P-ATP concentration required for a 50% decrease in open probability wa s 0.27 muM from the extracellular (cis) side and 20 muM from the cytop lasmic (trans) side. Comparison of the off rate constants revealed tha t TNP-ATP remained bound 28 times longer when added to the extracellul ar side compared with the cytoplasmic side. We performed competition s tudies to determine if TNP-ATP binds to the same sites as ATP. Additio n of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is th e result expected if opposite-sided TNP-ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nea rly 10-fold higher affinity for reconstituted rectifying colonic Cl- c hannels than any previously reported blocker (Singh, A. K., G. B. Afin k, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP shou ld be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification . In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state .