Venglarik C J, Singh A K, Wang R, Bridges R J
Department of Physiology and Biophysics, University of Alabama, Birmingham.
J Gen Physiol. 1993 Apr;101(4):545-69. doi: 10.1085/jgp.101.4.545.
Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular 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-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition 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 the result expected if opposite-sided TNP-ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should 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.
外向整流型30 - 50皮安的氯离子通道介导细胞容积调节和跨上皮运输。最近的几份报告表明，在细胞外浴液中添加ATP后，整流型氯离子通道会被阻断（奥尔顿，E.W.F.W.，S.D.曼宁，P.J.施拉特，D.M.格迪斯，和A.J.威廉姆斯。1991年。《生理学杂志》。443:137 - 159；保尔米希尔，M.，Y.李，K.维克曼，M.阿克曼，E.佩拉尔塔，和D.克拉彭。1992年。《自然》。356:238 - 241）。因此，我们决定使用一种亲和力更高的探针，对ATP结合位点进行更详细的研究。我们测试了ATP衍生物2'，3'，O -（2,4,6 - 三硝基环己二烯叉）腺苷5' - 三磷酸（TNP - ATP），它对某些核苷酸结合位点具有高亲和力。在此我们报告，当将TNP - ATP添加到任一侧浴液中时，它会阻断结肠氯离子通道，并且这种阻断与关闭 - 开放 - 阻断动力学模型一致。使开放概率降低50%所需的TNP - ATP浓度，从细胞外（顺式）侧为0.27微摩尔，从细胞质（反式）侧为20微摩尔。对解离速率常数的比较表明，与添加到细胞质侧相比，当添加到细胞外侧时，TNP - ATP的结合时间长28倍。我们进行了竞争研究，以确定TNP - ATP是否与ATP结合到相同位点。将ATP添加到含有TNP - ATP的同一浴液中，会降低通道幅度，并增加通道处于开放和快速阻断状态的时间（即爆发持续时间）。如果TNP - ATP和ATP竞争阻断，大概是通过结合到共同位点，那么这就是预期的结果。相反，将ATP添加到与含有TNP - ATP的一侧相对的浴液中，会降低幅度，但不会改变爆发持续时间。如果相对侧的TNP - ATP和ATP结合到不同位点，那么这就是预期的结果。总之，我们鉴定出一种ATP衍生物，它对重组的整流型结肠氯离子通道的亲和力比任何先前报道的阻断剂高近10倍（辛格，A.K.，G.B.阿芬克，C.J.文格拉里克，R.王，和R.J.布里奇斯。1991年。《美国生理学杂志》。260 [细胞生理学。30]：C51 - C63）。因此，TNP - ATP在未来离子通道核苷酸结合位点的研究中应该是有用的，并且可能在离子通道蛋白纯化的初步步骤中也有用。此外，我们已经获得了充分的证据，表明在结肠氯离子通道的相对两侧至少有两个核苷酸结合位点，并且占据任何一个位点都会产生阻断状态。
