Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers

Abstract

Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 μM Glyb and 200 μM Glip was 0.64 ± 0.03 (n = 7) and 0.48 ± 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 μM Tolb was 0.40 ± 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

“…Compounds that block anion conduction through CFTR channels (Sheppard and Robinson, 1997;Cai et al, 1999;Muanprasat et al, 2004;Sheppard, 2004;Zhang et al, 2004a;Sonawane et al, 2006Sonawane et al, , 2007de Hostos et al, 2011;Cui et al, 2012) may lead to treatments for secretory diarrhea and polycystic kidney disease. The CFTR-blocking small molecule 3-(3,5-dibromo-4-hydroxyphenyl)-N-(4-phenoxybenzyl)-1,2,4-oxadiazole-5-carboxamide (iOWH032), which currently is being studied in a clinical trial (de Hostos et al, 2011), is structurally related to the pore blocker N-(2-naphthalenyl)- [(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) (mol.…”

“…GlyH-101 carries a net negative charge (pK a ϭ 5.5) under physiological conditions (pH ϳ7.4) and is thought to block the CFTR by entering the channel from the extracellular side and binding to a site within the pore (Muanprasat et al, 2004;Sonawane et al, 2006Sonawane et al, , 2007. Although the mechanisms of action of pore blocking small molecules thought to act from the cytosolic side of the CFTR channel have been explored extensively in electrophysiological studies and amino acid substitution analyses (Sheppard and Robinson, 1997;Hwang and Sheppard, 1999;Zhou et al, 2002Zhou et al, , 2010Linsdell, 2005;Cui et al, 2012), the mechanism of action of GlyH-101 has been less well studied. We chose to investigate the binding of GlyH-101 because the open-channel blocker seemed likely to bind in a narrow part of the CFTR pore.…”

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

“…Based on functional data and homology models, CFTR has been predicted to contain five functional domains: two membranespanning domains (MSDs), each including six transmembrane (TM) helices; two nucleotide binding domains (NBD1 and NBD2); and a unique regulatory (R) domain, which carries multiple protein kinase A (PKA) consensus phosphorylation sites and is unique to CFTR in the ABC superfamily (1)(2)(3)(4)(5)(6). Functional studies from multiple groups have suggested that TM6 plays an essential role and TM12 contributes less to anion conduction and permeation properties in the CFTR channel pore (7)(8)(9)(10)(11).…”

“…We reported previously that human wild type CFTR (WT-CFTR) exhibits a predominant full open state with two rare subconductance states (s1 and s2). Infrequent subconductance behavior is seen in some CFTR mutants, such as T338A/C-and S1141A-CFTR (7,12). However, subconductance states are dominant events with short burst durations in CFTR channels bearing known salt bridge mutations, such as R352A, R347H, D993R, and D924R (13,14).…”

“…When arginine 334 at the extracellular end of TM6 is mutated to A or C, both mutants show s1 and s2 as the dominant open states with only brief transitions to the f state (15). Mutation D1152A in the inner vestibule of TM12 results in channels that frequently exhibit the s1 and s2 open states as well as the f open state (7). In the mutant channels exhibiting prominent subconductance states, the sequence of occupancy of the s1, s2, and f states does not appear to be random (12,16).…”