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Evidence for a superficial external blocker binding site in CFTR channel pore



Evidence for a superficial external blocker binding site in CFTR channel pore



Biophysical Journal 86(1): 332a, January



We have previously characterized a blocker binding site (Sint) accessible from the cytoplasmic side of the CFTR channel pore using hydrophobic aromatic compounds (Sohma et al. 2003. Biophy.J. 84:484a). We found that the smaller benzoic acid (BA) showed a unique block different from other larger aromatic compounds. We examined the blockade by BA and its derivatives in more detail, using a locked-open mutant CFTR, K1250A. In whole-cell experiments with a ramping pulse of +-200mV, BA inhibited whole-cell currents with a combination of two different voltage-dependent kinetics. The block was more prominent with hyperpolarization at negative Vm (Kd(0)=270 mM; zdelta=0.38) whereas much less voltage-dependent at Vm>+100 mV (Kd(0)=330 mM; zdelta<0.1). This result suggests two blocker binding sites, one accessible from the cytoplasmic side (Sint) and the other from the external side (Sext). Lowering external Cl- concentration from 154 mM to 24 mM potentiated the external BA block by 2-fold suggesting that Sext may be at the external entrance of the pore. Compared to BA, 4-Hydroxybenzoic acid (HBA), a more hydrophilic derivative, was less potent for the internal block (Kd(0)=1.8 M; zdelta=0.41) but was more potent for the external block (Kd=230 mM; zdelta=-0.09), further supporting the idea of two chemically distinct blocker binding sites. Preliminary data obtained from excised inside-out patches, showed that external application of HBA decreased single channel Cl- current with a minimal zdelta of -0.03 and Kd(0) of 300 mM. The fact that only small aromatic compounds exhibit this external block corroborates the idea that the CFTR pore has a stringent external entrance.

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Accession: 034887998

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