Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding
C J Venglarik1, A K Singh, R Wang
1Department of Physiology and Biophysics, University of Alabama, Birmingham.
Abstract:
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.
Insights
This study investigated the ATP binding site on colonic chloride channels using a novel ATP derivative, TNP-ATP. Results show TNP-ATP binds to at least two sites on the channel, aiding in understanding channel function and purification.
Area of Science:
- Molecular biology
- Ion channel physiology
- Biochemistry
Background:
- Outwardly rectifying chloride (Cl-) channels (30-50 pS) are crucial for cell volume regulation and transepithelial transport.
- Previous studies indicated ATP blocks these Cl- channels from the extracellular side.
Purpose of the Study:
- To investigate the ATP binding site on colonic Cl- channels using a high-affinity ATP derivative, 2',3',O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP).
- To characterize the binding kinetics and identify the location of nucleotide binding sites.
Main Methods:
- Utilized the ATP derivative TNP-ATP to probe the binding characteristics of colonic Cl- channels.
- Employed electrophysiological techniques to study channel blockade kinetics and ATP competition assays.
Main Results:
- TNP-ATP effectively blocked colonic Cl- channels from both extracellular and cytoplasmic sides, with higher affinity from the extracellular side (0.27 microM vs. 20 microM).
- TNP-ATP exhibited a 28-fold longer binding duration on the extracellular side compared to the cytoplasmic side.
- Competition studies with ATP suggested the presence of at least two distinct nucleotide binding sites on opposite sides of the channel.
Conclusions:
- TNP-ATP is a high-affinity blocker for colonic Cl- channels, approximately 10-fold more potent than previously identified blockers.
- Evidence supports the existence of at least two nucleotide binding sites on the colonic Cl- channel, located on opposite sides.
- TNP-ATP is a valuable tool for future research on ion channel nucleotide binding sites and potential protein purification.
Related Concept Videos
01:27ATP Driven Pumps I: An Overview
There are four main types of ATP-driven pumps - P-type, V-type, F-type, and ABC transporter. All these pumps are of varying complexities and are...
01:32Secondary Active Transport
01:48ATP Synthase: Mechanism
01:32Pinching-off of Coated Vesicles
01:19Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
01:19Membrane Asymmetry Regulating Transporters
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...