Metal Bridges Illuminate Transmembrane Domain Movements during Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Abstract

Background: Opening and closing of the CFTR channel involve conformational changes in the channel pore. Results: Different metal bridges stabilize the channel in the open or closed configuration. Conclusion: Opening and closing result from relative lateral movement of different transmembrane segments. Significance: This work defines the three-dimensional conformational changes underlying channel opening and closing.

Localizing a gate in CFTR

“…Although the exact gating motion of CFTR's TMDs remains unclear, recent studies do provide evidence for a significant rearrangement of different TMs (20,25) in addition to possible translational and rotational movements of individual TMs (15,16,46). Here, we propose that CFTR's pore-lining TMs may clog around the narrow region to craft a gate that ceases the chloride flow in the closed state.…”

“…Decades of biochemical and biophysical studies of CFTR have provided exquisite insights into not only how the molecular events in NBDs are coupled to opening and closing of the gate in TMDs (13) but also how different TMs craft CFTR's ion-conducting pore (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Specifically, studies using the substituted cysteine accessibility method (SCAM) have demonstrated that TM1, 3, 6, 9, 11, and 12 contribute to pore lining, with TM1, 6, and 12 intimately involved in gating motions of the TMDs (15,16,20,25). Although the molecular structure of CFTR's TMDs remains to be solved, both electrophysiological studies (15,20,21) and computational modeling (22,26,27) support the view that CFTR's pore is made up of a narrow tunnel flanked by an inner and an outer vestibule (Fig.…”

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

“…Initially, we investigated macroscopic currents carried by MTS-sensitive channel mutants after treatment with intracellular sodium pyrophosphate (PP i ; 2 mM) to stabilize the channel open state (34,35). We have previously used this approach in studies of state-dependent modification of cysteine residues introduced into Cys-less CFTR to isolate or enrich effects on open CFTR channels (27,36,37). Subsequently, effects of MTS modification on the rate of Cl…”

“…Channels were exposed to intracellular cysteine-reactive MTS reagents to covalently modify an introduced cysteine side chain. Two MTS reagents, the negatively charged [2-sulfonatoethyl]MTS (MTSES) and the positively charged [2-(trimethylammonium)ethyl]MTS (MTSET), were used at high concentrations (200 M) that we have previously shown to have no effect on Cys-less CFTR (22,(25)(26)(27). MTS reagents were applied to the cytoplasmic face of inside-out patches after stable current activation with PKA and ATP.…”

“…Pipette resistances were 1-2 megaohms for macroscopic current recording and 7-10 megaohms for single channel recording. Macroscopic current amplitudes were monitored during brief voltage ramps (to Ϯ50 mV) from a holding potential of 0 mV applied every 6 s as described in previous studies of modification of cysteine residues in CFTR (11,27,28). Channels were exposed to intracellular cysteine-reactive MTS reagents to covalently modify an introduced cysteine side chain.…”