Colonic Cl channel blockade by three classes of compounds

Abstract

We compared the potency and inhibitory actions of three different classes of organic acids on a Cl channel derived from colonic enterocyte plasma membrane vesicles. Chloride channels were incorporated into planar lipid bilayer membranes to examine the effects of the anthranilic acids, diphenylamine 2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), the indanyl alkanoic acids, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5-yl)oxy] acetic acid (IAA-94) and its stereoenantiomer IAA-95, and the disulfonic stilbene, 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS). Except for DNDS, each of the blockers was equipotent from both the outer membrane and the cytoplasmic side of the channel protein. The potency order from the outmembrane side was DNDS greater than IAA-94 = IAA-95 greater than NPPB much greater than DPC. In contrast, the potency order from the cytoplasmic side was IAA-94 = IAA-95 greater than NPPB greater than DNDS much greater than DPC. DPC and NPPB caused a concentration-dependent decrease in the single-channel conductance (fast block). DNDS, IAA-94, and IAA-95 caused a flickery-type block and a concentration-dependent decrease in open-channel probability. Kinetic analysis revealed that blockade could be explained by a linear closed-opened-blocked kinetic scheme. Similarities in the electrostatic potential maps of these open-channel blockers suggest they may bind to a single shared binding site within the channel protein.

Regulation of a Human Chloride Channel

“…This high conductance ORCC became active even when [Ca 2ϩ ] was buffered to 0.1 M (Fig. 2D) (23) and 100 M diphenylamine-2-carboxylic acid (24) are reported to inhibit ORCC in some cell preparations, these drugs did not prevent the appearance of ORCC in our experiments (data not shown).…”

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl– currents

“…The large Cl -currents in the presence of GST-syn 1A∆C were not affected by DIDS, which blocks a number of Cl -channels other than CFTR (26). Conversely, diphenylamine carboxylate (DPC), which does block CFTR Cl -channels (27), completely inhibited the currents that were potentiated by GST-syn 1A∆C ( Figure 5).…”

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

“…Single channel current records of rectifying colonic C1-channels in planar phospholipid bilayers were obtained as previously described (Reinhardt, Bridges, Rummel, and Lindemann, 1987;Bridges, Worrell, Frizzell, and Benos, 1989;Bridges and Benos, 1990;Singh et al, 1991). We used conventional polystyrene chambers for painted bilayers that were modified so the volume of each bath was 0.4 ml.…”

“…Having shown that TNP-ATP blocked the rectifying colonic C1-channel from either side, we performed event duration analysis to determine if TNP-ATP block con- closed time histograms obtained from current records before blocker addition could be described by single exponentials with an open time constant (%) of 270 -20 ms and a closed time constant (re) of ~3.0 ms (Reinhardt et al, 1987;Bridges et al, 1989;Singh et al, 1991 Second, % l should vary as a linear function of the blocker concentration with a slope corresponding to the on rate constant (kon). Third, the blocker should produce an additional exponential distribution in the closed time histograms corresponding to the blocked time constant ('rB).…”

“…In addition to their medium conductance in symmetric solutions containing 140 mM CI-(i.e., 30-50 pS) and characteristic outwardly rectifying current-voltage relation, these channels can be distinguished from other anion-selective channels based on their sensitivity to disulfonic stilbenes, anthranilic acids, and indanyloxyacetic acids (Lukfics and Moczydowlowski, 1990;Alton, Manning, Schlatter, Geddes, and Williams, 1991;Singh, Afink, Venglarik, Wang, and Bridges, 1991 ;Solc and Wine, 1991 ;Paulmichl, Li, Wickman, Ackerman, Peralta, and Clapham, 1992). Patch-clamp studies have also revealed that rectifying CI-channels can be activated by protein kinase (PK) and ATP in excised apical membrane patches from normal but not cystic fibrosis (CF) airway epithelial cells (Schoumacher, Shoemaker, Halm, Tallant, Wallace, and Frizzell, 1987;Li, McCann, Leidtke, Nairn, Greengard, and Welsh, 1988).…”