Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites.

Abstract

Outwardly rectifying 30-50-pS C1-channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying CI-channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. 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',0-(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 C1-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 I~M from the extracellular (c/s) side and 20 ~M 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 ifTNP-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 C1-channels than any previously reported blocker

Structure-Function Relation of a Cloned Epithelial Chloride Channel

“…A putative nucleotide binding site is located in the area of the outer mouth of the predicted pore. In fact, mutating this binding region from GxGxG to AxGxG or AxAxA (G = gly cine; A = alanine; x = different amino acids) dramatically reduces the ability of cAMP to block Itm-Nucleotide-sensitive chloride chan nels have also been found in tracheal epithelia [9], cardiac cells after swelling [10] and colonic chloride channels reconstituted in planar phospholipid bilayers [11], In contrast to the wild type In", the AxAxA mutant was highly sensitive to extracellular calcium concentration showing a dramatic de crease of the current amplitude in the nominal absence of calcium in the extracellular fluid (omitting calcium and adding 1 nuWEGTA). Furthermore, the kinetics of activation were characteristically changed: whereas the kinet ics of the wild type In,, comprised a fast activa tion followed by a slow inactivation at holding potentials more positive than +40 mV, the mutant current showed an initial fast activa tion followed by a further activation not sat urated after 800 ms.…”

Purinergic signalling in the gastrointestinal tract and related organs in health and disease

“…In a later abstract, this group reported that luminal ATP induces K + secretion via a P2Y 2 receptor in rat distal colonic mucosa [385]. It is interesting that TNP-ATP, which has since been identified as a potent antagonist at P2X1 and P2X3 receptors [686], was shown to block colonic Cl − channels [682], although there do not appear to be reports of the effects of TNP-ATP on P2Y receptors. In situ hybridisation studies have shown that the mRNA for P2X4 receptors is localised in rat intestinal crypts [653].…”

Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels

“…First, we measured the mean amplitude of control (icontr.) and fast-blocked channels (Oblo,k) from amplitude histograms as described previously (Venglarik et al, 1993b). The ratio of iblock'icontrol is related to the underlying change in P.…”