Non-Equilibrium Gating of CFTR Revealed by Nitrate as Charge Carriers

Abstract

CFTR, a member of the ATP-binding cassette protein superfamily, is a phosphorylation-activated but ATP-gated chloride channel. Like other anion channels, CFTR's pore is permeable to a wide variety of anions, including bromide, nitrate, iodide and bicarbonate. From the shift of reversal potentials under bi-ionic conditions for chloride, bromide and nitrate, we obtained a permeability sequence: NO3- > Br- > Cl-, a result consistent with previous reports, but the macroscopic conductance sequence, NO3- > Br- > Cl-, contradicts previously published Cl- > NO3- > Br-. Nonetheless, single-channel studies reveal a conductance sequence of Cl- > NO3- > Br-, suggesting bromide and nitrate may affect CFTR gating. By analyzing single channel kinetics, we found that NO3- indeed increases the open probability (0.71±0.01 versus 0.51±0.02 with Cl-) by increasing the opening rate and prolonging the open time. Interestingly, when examining recordings from patches containing one single channel in nitrate-based bath, we observed two distinct open-channel conductance levels (the smaller O1 state and the larger O2 state), a phenomenon similar to the effect of the R352C mutation on CFTR. Furthermore, statistical analysis of the pattern of gating transitions also reveals a prevalent transition of C→O1→O2→ C (46.3%) over C→O2→O1→ C (< 5%), a telltale sign for a violation of detailed balance and hence demanding an input of free energy to drive the gating transition in a preferred direction. In addition, a considerable fraction of openings contain more than one O1→O2 transition (38%), supporting the idea that more than one ATP molecule is hydrolyzed within an opening burst. Overall our studies indicate that nitrate, as a charge carrier, can be a new tool to probe CFTR's non-equilibrium gating cycle.