ABCMdb

PMID: 19448737

Zhou JJ, Linsdell P
Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties.
Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)4 2- ions. Login to comment 13 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Nous croyons que les mutations qui e &#b4;liminent les charges positives dans la quatrie `me boucle extracellulaire du CFTR (K892Q et R899Q) modifient significativement l`interaction entre les ions Pt(NO2)4 2- intracellulaires et extracellulaires. Login to comment 38 ABCC7 p.Arg334Gln Consistent with this distinction, we previously showed that the R334Q mutation at the outer mouth of the pore disrupted interactions between external Cl- and internal Pt(NO2)4 2-, without affecting interactions between external Pt(NO2)4 2- and internal Pt(NO2)4 2- (Zhou et al. 2007). Login to comment 63 ABCC7 p.Arg117Gln In each case, intracellular Pt(NO2)4 2- caused a voltage-dependent block with broadly similar affinity and voltage dependence (Fig. 1), although the apparent affinity of block was significantly increased in R117Q (Fig. 1D). Login to comment 67 ABCC7 p.Arg334Gln ABCC7 p.Lys335Ala ABCC7 p.Lys329Ala ABCC7 p.Arg1128Gln ABCC7 p.Lys114Cys Similar weakening of block by internal Pt(NO2)4 2-was also observed in K114C, K329A, K335A, and R1128Q (Fig. 2), as well as R334Q (Zhou et al. 2007). Login to comment 68 ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg899Gln In contrast, external Pt(NO2)4 2- ions did not significantly affect the apparent affinity of block by internal Pt(NO2)4 2- in R104Q, R117Q, or R899Q (Fig. 2). Login to comment 69 ABCC7 p.Lys892Gln Furthermore, in K892Q, the effect of external Pt(NO2)4 2-was reversed: in this mutant, block by internal Pt(NO2)4 2- ions was significantly strengthened by the presence of Pt(NO2)4 2- in the extracellular solution (Fig. 2). Login to comment 74 ABCC7 p.Lys892Glu ABCC7 p.Arg899Glu Moreover, reversal of the charge at these sites in ECL4 (in the K892E and R899E mutants) had no effect on unitary Cl-conductance in the presence of high symmetrical Cl- concentrations (Zhou et al. 2008), again consistent with these ECL4 residues not being involved directly in formation of the pore. Login to comment 75 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln To confirm that the K892Q and R899Q mutations did not directly alter pore properties, we further investigated interactions with external Pt(NO2)4 2- at the single-channel level (Fig. 3). Login to comment 77 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Furthermore, under these ionic conditions, voltage-dependent inhibition by extracellular Pt(NO2)4 2- ions in the extracellular solution appeared indistinguishable in wild type, K892Q, and R899Q (Figs. 3A, 3D, and 3E). Login to comment 80 ABCC7 p.Lys335Ala ABCC7 p.Arg117Gln ABCC7 p.Arg899Gln Nevertheless, the apparent Cl- dependence of block was significantly reduced in both K335A and R899Q, and significantly enhanced in R117Q (Fig. 4D). Login to comment 89 ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln (C) Mean fraction of control current remaining (I / I0) at different voltages after the addition of Pt(NO2)4 2- in wild type (*), R104Q (*), and R117Q (!). Login to comment 90 ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln Curves were fitted to mean data by eq. 1, giving Kd(0) = 89.5 mmol/L and -zd = -0.270 for wild type, Kd(0) = 131.1 mmol/L and -zd = -0.261 for R104Q, and Kd(0) = 44.8 mmol/L and -zd = -0.222 for R117Q. Login to comment 97 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Our present results suggest that this interaction is dependent on the presence of positive charges in ECL4 (K892, R899), since neutralization of these charges removes (R899Q) or reverses (K892Q) the effect of external Pt(NO2)4 2- ions on the blocking effect of internal Pt(NO2)4 2- (Fig. 2). Login to comment 100 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Thus, charge-neutralizing mutations K892Q and R899Q have no effect on unitary conductance (Fig. 3), as we have previously reported for the charge-re- Fig. 2. Login to comment 104 ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Curves were fitted to mean data by eq. 1, giving the following values: for wild type, (*) Kd(0) = 89.5 mmol/L and -zd = -0.270, (*) Kd(0) = 274.1 mmol/L and -zd = -0.262; for R104Q, (*) Kd(0) = 131.1 mmol/L and -zd = -0.261, (*) Kd(0) = 200.41 mmol/L and -zd = -0.326; for R117Q, (*) Kd(0) = 44.8 mmol/L and -zd = -0.222, (*) Kd(0) = 65.0 mmol/L and -zd = -0.304; for K892Q, (*) Kd(0) = 58.4 mmol/L and -zd = -0.275, (*) Kd(0) = 21.1 mmol/L and -zd = -0.250; and for R899Q, (*) Kd(0) = 102.8 mmol/L and -zd = -0.312, (*) Kd(0) = 92.8 mmol/L and -zd = -0.334. Login to comment 109 ABCC7 p.Lys892Glu ABCC7 p.Arg899Glu Zhou and Linsdell 391 versing mutations K892E and R899E under different ionic conditions (Zhou et al. 2008). Login to comment 110 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Furthermore, inhibition of Cl-current by extracellular Pt(NO2)4 2- ions-probably a pore-mediated effect (see above)-is unaltered in K892Q or R899Q (Fig. 3), suggesting these mutations do not directly affect extracellular Pt(NO2)4 2- interactions with the pore. Login to comment 111 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln We believe the effects of K892Q and R899Q on interactions between extracellular and intracellular Pt(NO2)4 2- ions shown in Fig. 2 therefore reflect interactions between extracellular Pt(NO2)4 2- ions and the extracellular-facing part of the CFTR protein at some distance from the pore. Login to comment 117 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln (B) Control single-channel i-V relationship for wild type (*), K892Q (&), and R899Q (!). Login to comment 121 ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln ABCC7 p.Lys892Gln In each case data were fitted by eq. 1, giving Kd (at 0 mV) of 5.98 mmol/L for wild type, 5.74 mmol/L for K892Q, and 5.33 mmol/L for R899Q, and zd of -0.382 for wild type, -0.390 for K892Q, and -0.434 for R899Q. Login to comment 127 ABCC7 p.Arg334Gln ABCC7 p.Lys335Ala ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln The effects of external Pt(NO2)4 2are sensitive to mutations away from the pore (K892Q, R899Q) but not deep within the pore (K335A, R334Q), which would be expected to alter ion-ion interactions in the pore. Login to comment 136 ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln For 154 mmol/L Cl- (*), fitted curves gave the following values: Kd(0) = 308.8 mmol/L and -zd = -0.441 for wild type; Kd(0) = 356.4 mmol/L and -zd = -0.378 for R104Q; Kd(0) = 234.9 mmol/L and -zd = -0.376 for R117Q; Kd(0) = 204.2 mmol/L and -zd = -0.395 for K892Q; and Kd(0) = 169.4 mmol/L and -zd = -0.462 for R899Q. Login to comment 143 ABCC7 p.Arg899Gln Nevertheless, in R899Q there is a significant decrease in the ability of external Cl-ions to antagonize block by internal Pt(NO2)4 2- (Fig. 4), suggesting that at least some part of the effect of external Cl-ions is mediated by a non-pore-dependent mechanism involving ECL4. Login to comment 145 ABCC7 p.Lys335Ala Interactions between external Cl- and internal Pt(NO2)4 2are also weakened in K335A (Fig. 4D), probably as a result of a decrease in direct pore-mediated interactions in this low-conductance mutant. Login to comment 146 ABCC7 p.Arg117Gln R117Q shows apparently increased sensitivity of internal Pt(NO2)4 2- block to external Cl-ions (Fig. 4D); however, this appears to reflect strong block under low-Cl- conditions (Fig. 2) more than weak block under high-Cl- conditions (Fig. 4). Login to comment 154 ABCC7 p.Arg117Gln ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg104Gln Thus, whereas direct pore block by externally applied Pt(NO2)4 2- ions is weakened in R104Q but not in R117Q (Zhou et al. 2008), loss of the interactions with external Pt(NO2)4 2- that influence the blocking effects of internal Pt(NO2)4 2- appears similar in both R104Q and R117Q (Fig. 2). Login to comment