Mechanism of lonidamine inhibition of the CFTR chloride channel

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Abstract
1. The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. 2. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage-dependent block of CFTR currents, with an apparent K(d) of 58 micro M at -100 mV. 3. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl(-) concentration. 4. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 micro M(-1) s(-1) and an off-rate of 143 s(-1). 5. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. 6. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action.