Abstract GlyH‐101 is a commonly used pore blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) to confirm the functional role of CFTR in model systems. Unlike most other anionic CFTR blockers GlyH‐101 blocks CFTR from the extracellular side, albeit previous studies suggesting an additional internal binding site. To explore the detailed mechanism of GlyH‐101 block we first examined GlyH‐101's effects on a hydrolysis‐deficient CFTR mutant (E1371S) whose open probability approaches unity. Whole‐cell recordings with extracellularly applied GlyH‐101 revealed two phases of current reduction: a rapid initial drop within seconds and a slower decay over tens of seconds. The fast phase blockade exhibited voltage dependence, consistent with previously reported external pore blocking. Single‐channel recordings in excised inside‐out patches with GlyH‐101 in the pipette solution showed a voltage‐dependent blockade with |zδ| of 0.38, close to that reported previously. However intracellular application of GlyH‐101 also induced a similar voltage‐dependent block, suggesting membrane permeation and subsequent development of external block. To decrease the membrane permeability of GlyH‐101 we synthesised a hydrophilic analogue GlyH‐101‐1. Application of GlyH‐101‐1 from the cytoplasmic side of the membrane induced voltage‐independent CFTR current inhibition. Single‐channel recordings revealed two distinct shut states in the presence of cytoplasmic GlyH‐101‐1, consistent with the two‐step blocking kinetics previously described for CFTRinh‐172. Single‐channel kinetics with a hydrophilic CFTRinh‐172 analogue further confirmed this inhibitory mechanism. Taken together our findings establish two distinct inhibitory mechanisms for GlyH‐101: a voltage‐dependent on‐off block through the external entrance and a two‐step voltage‐independent inhibition through the internal entrance. image Key points GlyH‐101, a hydrophobic cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, can diffuse across cell membranes and plug CFTR's pore from both external and internal ends. Although the external block is voltage dependent, the internal block is not. A hydrophilic GlyH‐101 derivative is synthesized and shown to block CFTR's pore from its internal entrance in a voltage‐independent manner. At the single‐channel level our data support a two‐step inhibitory mechanism: a fast binding of the blocker in the pore and a slow conformational change step following binding – a mechanism first proposed for CFTRinh‐172. Although CFTRinh‐172 itself did not show two‐step inhibitory action, leveraging the cryo‐EM structure of CFTR/CFTRinh‐172 complex, we synthesized an analogue that indeed blocks CFTR through the same two‐step mechanism. Our studies uncovered a common mechanism for CFTR inhibitors and underscore the potential role of structure‐based drug design in developing drugs for diseases caused by hyperactive CFTR.
Chang et al. (Thu,) studied this question.