ABCMdb

PMID: 25673337

Rubaiy HN, Linsdell P
Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12., [PubMed]

Sentences

No. Mutations Sentence Comment

32 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala Specifically, mutations that reduce side-chain volume (F337A, F337S) disrupt the relationship between the anion permeability and anion free energy of hydration [18]. Login to comment 41 ABCC7 p.Phe337Ala ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln In contrast, the F337A mutation disrupts the normal Fig. 1 Block by intracellular Au(CN)2 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of Au(CN)2 - to the intracellular (bath) solution at the concentrations stated. Login to comment 48 ABCC7 p.Glu1371Gln In order to isolate anion effects on Cl-permeation from effects on channel gating [17, 24], experiments were carried out on constitutively active E1371Q-CFTR channels [22, 24]. Login to comment 49 ABCC7 p.Glu1371Gln Additional mutations were introduced into this E1371Q background using the QuikChange site-directed mutagenesis system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing. Login to comment 50 ABCC7 p.Glu1371Gln Macroscopic E1371Q-CFTR current-voltage (I-V) relationships were recorded using depolarizing voltage ramps during patch clamp recordings from inside-out membrane patches, as described in detail recently [24]. Login to comment 57 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln The macroscopic current reversal potential (VREV) Fig. 2 Block by intracellular SCN- and C(CN)3 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of 10 mM SCN- to the intracellular (bath) solution. c Mean KD values for SCN- block for these channel constructs, obtained as described for Au(CN)2 - in Fig. 1. Login to comment 68 ABCC7 p.Glu1371Gln Results Intracellular Au(CN)2 - ions cause a voltage-dependent block of open CFTR channel pores [17], a finding that is recapitulated in constitutively open E1371Q channels (Fig. 1). Login to comment 71 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln Thus, the mean KD was increased between 92-fold (at -100 mV) and 19-fold (at ?60 mV) in K95Q/E1371Q (Fig. 1d). Login to comment 72 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln Similar weakening of block in K95Q/E1371Q Fig. 3 Strength of Au(CN)2 - block is dependent on the number of positive charges in the pore inner vestibule. Login to comment 73 ABCC7 p.Glu1371Gln ABCC7 p.Lys95His a Example macroscopic I-V relationships for K95H/E1371Q CFTR channels recorded using bath solutions at pH 5.5 (left) or pH 9.0 (right, different patch). Login to comment 75 ABCC7 p.Glu1371Gln ABCC7 p.Lys95His b Mean KD values for Au(CN)2 - block of K95H/E1371Q at these two different pHs, obtained as described for Au(CN)2 - in Fig. 1. Login to comment 77 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Lys95His c Relationship between the observed KD values for Au(CN)2 - block (at -100 mV) and bath solution pH in K95H/ E1371Q and E1371Q. Login to comment 78 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys d Example macroscopic I-V relationships for I344K/E1371Q (left) and K95Q/I344K/E1371Q (right) CFTR channels recorded before (control) and after the addition of a low concentration (10 lM) of Au(CN)2 - to the intracellular (bath) solution. Login to comment 79 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys ABCC7 p.Val345Lys e Mean KD values for Au(CN)2 - block for these channel constructs, as well as the additional positive charge mutants V345K/ E1371Q and S1141K/E1371Q, obtained as described in Fig. 1. f Relationship between the observed KD values for Au(CN)2 - block (at -100 mV) and the expected number of fixed positive charges in the pore inner vestibule in different channel constructs. Login to comment 80 ABCC7 p.Lys95His K95H0 refers to the unprotonated form of the histidine side chain (as expected at pH 9.0) and K95H? Login to comment 82 ABCC7 p.Glu1371Gln Asterisks indicate a significant difference from E1371Q (*P \ 0.05, **P \ 0.0001). Login to comment 86 ABCC7 p.Glu1371Gln ABCC7 p.Lys95His As shown in Fig. 3a and b, block of K95H/E1371Q by intracellular Au(CN)2 - was drastically stronger at pH 5.5 compared to pH 9.0, with the mean KD at -100 mV being increased 244-fold at the more alkaline pH. Login to comment 87 ABCC7 p.Glu1371Gln In contrast, Au(CN)2 - block of E1371Q was not pH-dependent (Fig. 3c). Login to comment 90 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys ABCC7 p.Val345Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Consistent with this, mutation of any of these three residues to lysine (I344K, V345K, S1141K) led to a significant strengthening of Au(CN)2 - block (Fig. 3d-f), with mean KD values at -100 mV being reduced by 18-fold (I344K/E1371Q), 17-fold (V345K/E1371Q) and 7-fold (S1141K/E1371Q), respectively. Login to comment 91 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys ''Moving`` the key positive charge from TM1 to TM6 by mutagenesis (in the K95Q/I344K/E1371Q mutant) resulted in Au(CN)2 - block that was intermediate in strength between E1371Q and I344K/E1371Q (Fig. 3e-f). Login to comment 93 ABCC7 p.Phe337Ala ABCC7 p.Glu1371Gln As well as showing tight binding of lyotropic permeant anions, CFTR also shows a lyotropic anion permeability Fig. 4 Block of F337A/ E1371Q channels by intracellular lyotropic permeant anions. Login to comment 94 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln a, b Example macroscopic I-V relationships for F337A/E1371Q CFTR channels recorded before (control) and after the addition of Au(CN)2 - (1 mM) or SCN- (10 mM) to the intracellular (bath) solution. c Mean KD values for Au(CN)2 - , SCN- , and C(CN)3 - (estimated at -100 mV as described in Figs. 1 and 2) compared in E1371Q, K95Q/ E1371Q, and F337A/E1371Q. Login to comment 95 ABCC7 p.Glu1371Gln Asterisks indicate a significant difference from E1371Q (P \ 0.01). Login to comment 97 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala This permeability sequence is disrupted by mutations at the putative narrow region of the pore, located more extracellularly in the pore than K95, in particular F337A and F337S (see ''Introduction``). Login to comment 98 ABCC7 p.Phe337Ala ABCC7 p.Lys95Gln As shown in Fig. 4, the F337A mutation had only a minor effect on binding of lyotropic Au(CN)2 - , SCN- and C(CN)3 - ions when compared to the K95Q mutation, suggesting that these anions can still bind relatively tightly in the pore even when lyotropic permeability selectivity is compromised. Login to comment 100 ABCC7 p.Glu1371Gln As shown in Fig. 5, macroscopic current reversal potential measurements with different anions present in the intracellular solution gave a permeability selectivity sequence SCN- [ NO3 - [ Br- [ Cl- [ F- for E1371Q-CFTR, with very similar relative permeabilities for those previously reported for wild type CFTR under similar conditions [11]. Login to comment 101 ABCC7 p.Phe337Ala ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Consistent with the proposed role of F337 in controlling lyotropic anion permeability, the permeability of all anions tested in F337A/E1371Q was significantly changed relative to E1371Q (Fig. 5b), with the permeability selectivity sequence being changed to NO3 - - C SCN- C Cl- [ Br- [ F- . Login to comment 102 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Glu1371Gln Again, relative permeability values for F337A/E1371Q were similar to those reported previously for F337A [18]. Login to comment 103 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys In contrast, neither K95Q nor I344K altered the permeability selectivity sequence; in fact, the only significant difference between either K95Q/ E1371Q or I344K/E1371Q compared to E1371Q was a small increase in PF/PCl in K95Q/E1371Q. Login to comment 106 ABCC7 p.Phe337Ala ABCC7 p.Glu1371Gln Note that the range of current reversal potentials was greatly reduced in F337A/E1371Q, suggesting a relative loss of permeability selectivity in this mutant. Login to comment 107 ABCC7 p.Glu1371Gln b Mean PX/PCl values calculated from current reversal potential measurements under these conditions as described in ''Materials and methods.`` Asterisks indicate a significant difference from E1371Q (*P \ 0.05; **P \ 0.002). Login to comment 109 ABCC7 p.Phe337Ala ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Note that the normal lyotropic relationship between relative permeability and Gh is greatly reduced in F337A/E1371Q but retained in K95Q/E1371Q and I344K/E1371Q. Login to comment 112 ABCC7 p.Lys95Gln ABCC7 p.Lys95His Removal of a positive charge in the pore inner vestibule by mutagenesis (in the K95Q mutant) or by increasing pH (in K95H) dramatically increases the apparent KD (Figs. 1, 2, 3, 4), suggesting that this positive charge is required for tight binding of permeant anions. Login to comment 113 ABCC7 p.Lys95Gln ABCC7 p.Lys95His In fact, since Au(CN)2 - , SCN- and C(CN)3 - are permeant anions and presumably have access to the entire pore, it is possible that the residual block observed in K95Q and in K95H (at pH 9.0) reflects interactions with a different part of the pore. Login to comment 118 ABCC7 p.Lys95Gln Thus, mutations that dramatically decrease (K95Q) or increase (I344 K) the strength of permeant anion binding inside the pore had only minimal effects on anion relative permeability (Fig. 5). Login to comment 120 ABCC7 p.Phe337Ala Conversely, a mutation that is known to have a strong effect on relative permeability-F337A (Fig. 5)-had relatively minor effects on permeant anion binding (Fig. 4). Login to comment