ABCMdb

ABCC7 p.Arg303Gln

None has been submitted yet.

Publications

PMID: 17043152 [PubMed] Aubin CN et al: "Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel."

No. Sentence Comment

66 To investigate the role of the charge on these residues in controlling I-V shape, these two arginines were also mutated to neutral glutamine residues; as shown in Fig. 3, R303Q and R352Q were also associated with outward rectification, although in both cases the degree of rectification observed was significantly less than with the charge-changing glutamate substitutions. Login to comment
71 As shown in Fig. 3 B, macroscopic I-V rectification was indeed highly sensitive to symmetrical Cl-concentration in both R303E and R303Q, suggesting that increasing the Cl-concentration partially reverses the effects of removing these positive charges. Login to comment
75 (B) The degree of rectification in R303E, R303Q, and R352Q is dependent on the Cl- concentra- tion. Login to comment
125 These changes in I-V shape were not observed in R303Q (unpublished data), suggesting that they are specific for the introduction of a reactive cysteine at this position. Login to comment
184 Furthermore, rectification in R303Q and R303E appeared more sensitive to the intracellular Cl-concentration than in R352Q and R352E (Fig. 3 B). Login to comment
208 R134Q is associated with outward rectification (Fig. 10, A and B); however, this effect shows a complex Cl-dependence that, unlike that of R303E, R303Q, and R352Q (Fig. 3 B), is not consistent with a simple surface charge effect. Furthermore, R134Q shows an extremely small apparent unitary conductance (Fig. 10 C). Login to comment

PMID: 17293558 [PubMed] St Aubin CN et al: "Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

95 As shown in Fig. 5, a mutation that removed one of these positive charges, R303Q, significantly weakened the inhibitory effects of suramin. Login to comment
105 A, example of leak-subtracted current-voltage relationships for K95Q and R303Q-CFTR recorded before (control) and after (ϩ suramin) the addition of 10 ␮M suramin to the intracellular solution. Login to comment
106 B, mean fraction of control current remaining (I/I0) after the addition of different concentrations of suramin at a membrane potential of -100 mV in K95Q (F) and R303Q (E). Login to comment
107 Mean of data from five to six patches is shown, fitted by eq. 1 (solid lines), giving Kd values of 11.6 ␮M for K95Q and 48.2 ␮M for R303Q. Login to comment
115 Figure 7 compares the blocking effects of intracellular glibenclamide in wild-type CFTR with the charge-neutralizing mutants R303Q and K95Q. Login to comment
116 It can be seen that glibenclamide inhibition is weakened in R303Q, although the effects of this mutant are not as dramatic as those seen in K95Q. Login to comment
117 Furthermore, a double mutant in which both of these positively charged residues were mutated to neutral glutamines (K95Q/R303Q) showed glibenclamide sensitivity similar to that of K95Q alone (Fig. 7). Login to comment
119 As shown in Fig. 8, DNDS block was also weakened in R303Q; however, this mutation did not significantly affect block by lonidamine, NPPB, or TLCS. Login to comment
120 The overall effects of the R303Q mutation on the affinity of block by the six different substances used in the present study are summarized in Fig. 9 and Table 1; the structures of these six substances are shown in Fig. 1. Login to comment
128 A, example of leak-subtracted current-voltage relationships for wild-type, R303Q, K95Q, and the K95Q/R303Q double mutant recorded before (control) and after (ϩ glibenclamide) the addition of 30 ␮M glibenclamide to the intracellular solution. Login to comment
142 Inhibition of R303Q-CFTR by open-channel blockers. Login to comment
143 A, example of leak-subtracted current-voltage relationships for wild type and R303Q-CFTR recorded before (control) and after the addition of the named blocker to the intracellular solution: DNDS (100 ␮M), lonidamine (100 ␮M), NPPB (50 ␮M), or TLCS (50 ␮M). Login to comment
144 B, mean fraction of control current remaining (I/I0) after the addition of these concentrations of blockers in wild type (E) and R303Q (F). Login to comment
159 Mean Kd values estimated at -100 mV for each of the six CFTR inhibitors used in the present study under identical ionic conditions for wild type (Ⅺ) and R303Q (f). Login to comment
164 Blocker K95Q R303Q Suramin 0 ϩϩ Glibenclamide ϩϩ ϩ DNDS ϩϩ ϩ Lonidamine ϩϩ 0 NPPB ϩϩ 0 TLCS ϩϩ 0 0, Ͻ2-fold change in Kd; ϩ, 2to 8-fold increase in Kd; ϩϩ, Ͼ8-fold increase in Kd. Login to comment
176 The charge-neutralizing R303Q mutation greatly weakens suramin block, significantly weakens the blocking effects of glibenclamide and DNDS (but to a lesser extent than for suramin), and has no apparent effect on block by lonidamine, NPPB, or TLCS (Fig. 9 and Table 1). Login to comment
178 The reasons for the minor effect of the R303Q mutation on inhibition by glibenclamide and DNDS are less clear. Login to comment
181 Removal of this surface charge in R303Q would then reduce the rate of glibenclamide and DNDS entry into the pore and to their primary binding site near Lys95. Login to comment
190 Whatever the mechanism, the far greater effects of the K95Q mutation on block by glibenclamide and DNDS relative to R303Q (Table 1) are consistent with the most important interaction underlying open-channel block by these two molecules being with Lys95. Login to comment

PMID: 17673962 [PubMed] Zhou JJ et al: "Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

68 Whether studied under conditions of low (Fig. 1a) or high (Fig. 1b) extracellular ClÀ concentration, Pt(NO2)4 2À block is significantly weakened in both K95Q and R303Q (Figs. 1c, 2). Login to comment
70 Furthermore, a double mutant in which both of these positive charges are neutralized (K95Q/R303Q) generated currents that were almost completely insensitive to the blocking effects of Pt(NO2)4 2À (Fig. 1), consistent with the overall channel-blocking effects of Pt(NO2)4 2À ions being the result of interactions with both of these purportedly independent sites (St. Aubin et al., 2007). Login to comment
71 In spite of the weakened blocking effects of intracellular Pt(NO2)4 2À seen in both K95Q and R303Q, block of each of these two mutants was still significantly weakened by extracellular ClÀ ions (Figs. 1c, 2). Login to comment
74 However, unlike K95Q and R303Q, the R334Q mutant weakened Pt(NO2)4 2À block at low, but not high, extracellular ClÀ concentration (Fig. 2). Login to comment
76 As described above, weakened block by intracellular Pt(NO2)4 2À ions in K95Q and R303Q is consistent with Fig. 1 Removal of positive charges in the pore weakens block by intracellular Pt(NO2)4 2À ions. Login to comment
81 Mean of data from three to eight patches. Fitted lines are to equation 1, with the following parameters: wild type 4 mM external ClÀ , Kd(0) = 85.8 lM, zd = À0.201; wild type 154 mM external ClÀ , Kd(0) = 387 lM, zd = À0.344; K95Q 4 mM external ClÀ , Kd(0) = 403 lM, zd = À0.130; K95Q 154 mM external ClÀ , Kd(0) = 978 lM, zd = À0.227; R303Q 4 mM external ClÀ , Kd(0) = 300 lM, zd = À0.096; R303Q 154 mM external ClÀ , Kd(0) = 904 lM, zd = À0.197; R334Q 4 mM external ClÀ , Kd(0) = 286 lM, zd = À0.330; R334Q 154 mM external ClÀ , Kd(0) = 256 lM, zd = À0.307. Login to comment
82 Because block was so weak at this concentration, data for the K95Q/R303Q double mutant were not fitted current models of the pore that place these two positively charged amino acid residues within the pore inner vestibule. Login to comment
136 Example macroscopic current-voltage relationships for wild type and each R334 mutant studied, as well as mutations that neutralize positive charges involved in binding of intracellular Pt(NO2)4 2À (K95Q, R303Q), are compared in Figure 11a. Login to comment
140 Both K95Q and R303Q are associated with outward rectification under control conditions; this rectification was apparently weakened in the presence of extracellular Pt(NO2)4 2À , most likely due to voltage-dependent current inhibition, which was most prominent at depolarized voltages. Login to comment
141 The mean effects of 10 mM Pt(NO2)4 2À in each channel variant, analyzed as described above for wild type (Fig. 10c), are illustrated in Figure 11b (for R334 mutants) and 11c (for K95Q and R303Q). Login to comment
142 Considering only the data at +80 mV (Fig. 11d), where block of wild-type CFTR is strongest (Figs. 9, 10), the blocking effects of Pt(NO2)4 2À are slightly (but significantly) weakened in K95Q, R303Q and R334K (p < 0.05) but practically abolished in all other R334 mutants (p < 0.0005). Login to comment
143 In fact, only wild type, R334K, K95Q and R303Q - those mutants that retain a positive charge at position 334 - were significantly affected by 10 mM Pt(NO2)4 2À according to this analysis (as illustrated by the daggers in Fig. 11d, p < 0.001), whereas all mutants associated with removal of the positive charge at R334 showed no significant differences in the absence or presence of external Pt(NO2)4 2À (p > 0.15). Login to comment
149 Our present results suggest that block by intracellular Pt(NO2)4 2À involves interactions with positively charged amino acid side chains in the wide inner vestibule of the pore since mutations that remove these positive charges (K95Q, R303Q) lead to significant weakening of Pt(NO2)4 2À block (Figs. 1, 2). Login to comment
150 Furthermore, removal of both positive charges (in the K95Q/R303Q double mutant) gave rise to a channel that was almost completely resistant to the blocking effects of internal Pt(NO2)4 2À (Fig. 1), suggesting that both positive charges contribute to Pt(NO2)4 2À binding in the pore inner vestibule. Login to comment
178 Thus, all R334 mutants studied disrupted the dependence of intracellular Pt(NO2)4 2À block on extracellular ClÀ ions (Fig. 5), whereas Pt(NO2)4 2À block of both K95Q and R303Q remained Cl-dependent (Figs. 1, 2). Login to comment
226 Note the change in current rectification induced by extracellular Pt(NO2)4 2À in wild type, R334K, K95Q and R303Q but not other R334 mutants. Login to comment
228 ), R334E (5), R334H (j), R334K (), R334L (h), R334Q (u); c wild type (d), K95Q (m), R303Q (Å). Login to comment

PMID: 20926782 [PubMed] Li MS et al: "Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate."

No. Sentence Comment

118 To investigate whether these positively charged residues influence the strength of channel block by cytosolic anions in intact cells, we neutralized these charges in an E1371Q background. Login to comment
119 Figure 6A shows macroscopic currents carried by K95Q/ E1371Q-, R303Q/E1371Q-, and K978Q/E1371Q-CFTR in Fig. 4. Login to comment
130 Comparison of the fractional current in cell-attached patches seen in these mutants with those for E1371Q under the same ionic conditions (Fig. 1) suggests that block of each of these mutants is weakened under cell-attached conditions, particularly at hyperpolarized voltages, where block is usually strongest (Fig. 6B). Login to comment
131 Interestingly, the effects of the K95Q and R303Q mutations did not appear additive, with the K95Q/R303Q/E1371Q mutant showing relief of block following patch excision similar to that shown by K95Q/E1371Q (Fig. 6). Login to comment

PMID: 19019741 [PubMed] Tang L et al: "Mechanism of direct bicarbonate transport by the CFTR anion channel."

No. Sentence Comment

94 Fig. 4 shows that these same charge-neutralizing mutations (K95Q, R303Q) also drastically reduce the ability of these inhibitors to block HCO3 - currents under the same ionic conditions used in Fig. 3 (50 mM HCO3 - intracellular, 50 mM gluconate extracellular). Login to comment
96 Another mutation, R303Q, greatly weakened the blocking effects of suramin on HCO3 - currents. Login to comment
140 Mutations in the pore decrease the blocker sensitivity of bicarbonate currents (A) Example leak-subtracted I-V relationship recorded under the same ionic conditions used in Fig. 3B in K95Q-CFTR (left, centre panels) or R303Q-CFTR (right panel). Login to comment

PMID: 17397825 [PubMed] Zhou JJ et al: "Molecular mechanism of arachidonic acid inhibition of the CFTR chloride channel."

No. Sentence Comment

2 Charge-neutralizing mutants K95Q and R303Q both increased the Kd for inhibition from ~3.5 bc;M in wild type to ~17 bc;M. Login to comment
35 As also shown in Fig. 1, inhibition by arachidonic acid is dramatically weakened by point mutations which remove important positive charges from the pore inner vestibule, K95Q and R303Q, leading to approximately a 5-fold increase in Kd in both cases. Login to comment
44 (A) Example leak-subtracted current-voltage relationships recorded from inside-out membrane patches for wild type, K95Q, and R303Q-CFTR. Login to comment
46 (B) Mean fraction of control current remaining (I/I0) after the addition of different concentrations of arachidonic acid at a membrane potential of -100 mV, for wild type (cf;), K95Q (cb;) and R303Q (bc;). Login to comment
48 In each case the fitted lines are to Eq. (1), giving a Kd of 3.5 bc;M and nH of 1.54 for wild type, Kd of 17.2 bc;M and nH of 0.77 for K95Q, and Kd of 16.5 bc;M and nH of 1.06 for R303Q. Login to comment