High affinity ATP/ADP analogues as new tools for studying CFTR gating.
Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating.
Zhou
Zhen
Z
Department of Medical Pharmacology, University of Missouri-Columbia, Columbia, MO 65211, USA.
Wang
Xiaohui
X
Li
Min
M
Sohma
Yoshiro
Y
Zou
Xiaoqin
X
Hwang
Tzyh-Chang
TC
D018528:ATP-Binding Cassette Transporters / Q000502:physiology; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D005260:Female; D006868:Hydrolysis; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials; D009154:Mutation; D011485:Protein Binding
D018528:ATP-Binding Cassette Transporters; C507839:N6-(2-phenylethyl)-ATP; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate