Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C62
Location of the ATP-dependent gate in the cystic fibrosis transmembrane conductance regulator channel pore
W. Wang1, P. Linsdell1
1. Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to lead to opening of a single “gate” within the permeation pathway, however, the location of such a gate has not been described. Recently our group described the presence of a “barrier” within the inactive channel pore that prevents access from the cytoplasm to the narrow pore region (1). Here we present evidence that this barrier reflects the location of the channel gate. We used inside-out patch clamp recording from the BHK cell line to monitor access of cytosolic cysteine reactive reagents (MTSES and MTSET) to reporter cysteines introduced at different sites in a cysteine-less CFTR pore, located in transmembrane (TM) regions TM1 and TM6. To investigate the relationship between access to these sites and ATP-dependent channel gating, we used pharmacological and mutagenic approaches to manipulate NBD function. Addition of 2 mM pyrophosphate (PPi) to inhibit ATP hydrolysis, promote locking in the open state and increase overall channel open probability significantly increased the rate of modification by both MTSES and MTSET at Q98C (TM1) and I344C (TM6) (p<0.05) but had no effect on the rate of modification at K95C (TM1) or V345C (TM6) (p>0.4). We also manipulated NBD function by introducing the mutations K464A (which decreases channel opening rate and open probability) and E1371Q (which slows channel closure and increases open probability). The E1371Q mutation significantly increased the rate of MTSES modification at Q98C and I344C (p<0.02) but had no effect on the rate of modification at K95C or V345C (p>0.25). The K464A mutation significantly decreased the rate of MTSET modification at Q98C and I344C (p<0.005) but had no effect on the rate of modification at K95C or V345C (p>0.5). These results therefore suggest that altering NBD function pharmacologically (using PPi) or non-pharmacologically (by mutagenesis) reveals a positive relationship between channel open probability and accessibility of Q98C and I344C to cytoplasmic MTS reagents, whereas accessibility of K95C and V345C are unaffected by NBD-driven channel gating. Our results suggest that access from the cytoplasm to K95 and V345 is similar in open and in closed channels. In contrast, channel opening increases access to Q98 and I344, located further into the pore. We propose that the ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these amino acids.
Where applicable, experiments conform with Society ethical requirements