ABCMdb

PMID: 10385235

Walsh KB, Long KJ, Shen X
Structural and ionic determinants of 5-nitro-2-(3-phenylprophyl-amino)-benzoic acid block of the CFTR chloride channel.
Br J Pharmacol. 1999 May;127(2):369-76., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu 4 NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. Login to comment 6 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu However, the Kds for NPPB block were increased to 371 and 1573 mM, for the K335E and R347E mutants, respectively. Login to comment 26 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ), and the K335E and R347E mutants obtained from Dr K Kunzelmann (Albert-Ludwigs University, Freiburg, Germany). Login to comment 76 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu Eect of NPPB on K335E and R347E CFTR mutants The pKa of NPPB is close to 4.5 (Wangemann et al., 1986; Walsh & Wang, 1998), and thus the drug molecules are predominately charged (499%) in the ND-96 solution at pH 7.5. Login to comment 77 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu Since the negatively charged drug may interact with positively charged amino acid residues in the pore of the CFTR channel, we examined the eect of NPPB on the mutants K335E and R347E. Login to comment 90 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu The K335E channel displayed a more linear I/V relationship (Figure 5) and the R347E channel a more outward-rectifying I/V relationship (Figure 6) than that measured with the wild-type channel (Figure 3). Login to comment 92 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu NPPB was less eective in blocking the CFTR currents in oocytes expressing the K335E and R347E channels. Login to comment 93 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu The Kd for NPPB block of the current was increased from 166 mM for the wild-type to 371 and 1573 mM for the K335E and R347E mutants, respectively (Figures 5 and Figure 5 Eect of NPPB on the K335E CFTR channel. Left panel: I/V relationship for the CFTR current measured in the presence and absence of 100 mM NPPB. Login to comment 95 ABCC7 p.Lys335Glu Right panel: concentration versus response curve for inhibition of the wild-type and K335E channels by NPPB. Login to comment 98 ABCC7 p.Lys335Glu For the K335E data, each point represents the mean+s.e.mean of three to ®ve experiments. Login to comment 99 ABCC7 p.Lys335Glu The theoretical curve for the K335E data provided a Kd of 371 mM. Login to comment 100 ABCC7 p.Arg347Glu Figure 6 Eect of NPPB on the R347E CFTR channel. Left panel: I/V relationship for the CFTR current measured in the presence and absence of 100 mM NPPB. Login to comment 102 ABCC7 p.Arg347Glu Right panel: concentration versus response curve for inhibition of the wild-type and R347E channels by NPPB. Login to comment 105 ABCC7 p.Arg347Glu For the R347E data, each point represents the mean+s.e.mean of three to ®ve experiments. Login to comment 106 ABCC7 p.Arg347Glu The theoretical curve for the R347E data provided a Kd of 1573 mM. Login to comment 108 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu Although the sensitivity of the mutant channels to NPPB was reduced, both the K335E and R347E mutants displayed a voltage-dependence to NPPB block that was similar to the wild-type channel (Figure 7). Login to comment 109 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu The slopes of the lines obtained from the relationship between the fractional block (Id/Io) and voltage had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E). Login to comment 116 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu Relationship between the fractional drug block (Id/I0) and the membrane potential determined for the wild-type (100 mM NPPB), K335E (400 mM NPPB) and R347E (1 mM NPPB) CFTR channels. Login to comment 117 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu The slopes of the straight lines had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E). Login to comment 148 ABCC7 p.Arg347Asp Furthermore, the R347D construct lacks multi-ion pore behaviour; a property measured with the wild-type channel in mixtures of Cl7 and SCN7 (Tabcharani et al., 1993; Linsdell et al., 1997). Login to comment 149 ABCC7 p.Arg347Asp Unlike the wild-type channel, the R347D mutant is insensitive to block by high concentrations of internally applied 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) (Linsdell & Hanrahan, 1996a). Login to comment 152 ABCC7 p.Arg347Glu This insensitivity is most striking for the R347E mutant, suggesting that this positively charged residue serves as an important determinant in the binding of the negatively charged drug molecules. Login to comment 153 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu While the concentration versus response curves for NPPB block of mutants K335E and R347E were shifted to higher concentrations, NPPB produced a voltage-dependent block in the mutants that was almost identical to that of the wild-type channel. Login to comment 158 ABCC7 p.Ser341Ala Based on the ®nding that the mutant S341A displays a 5 fold reduction in DPC anity, it was suggested that DPC interacts through the hydroxyl group at this residue (McDonough et al., 1994). Login to comment 159 ABCC7 p.Arg347Glu Although the anity of the R347E mutant for DPC has not been determined, it is likely that mutations at several sites in the M6 region will eect arylaminobenzoate block. Login to comment 160 ABCC7 p.Lys335Glu This is supported by our observation that a mutation of K335, a site predicted to reside closer to the external mouth of the channel (Riordan et al., 1989), resulted in a small, but signi®cant change in NPPB potency (IC50=166 mM for wild-type and 371 mM for K335E). Login to comment 162 ABCC7 p.Arg347Asp Interestingly, this eect of SCN7 is absent in the R347D mutant (Tabcharani et al., 1993). Login to comment