ABCMdb

PMID: 7522483

McDonough S, Davidson N, Lester HA, McCarty NA
Novel pore-lining residues in CFTR that govern permeation and open-channel block.
Neuron. 1994 Sep;13(3):623-34., [PubMed]

Sentences

No. Mutations Sentence Comment

51 ABCC7 p.Ser341Ala Figure 3C shows data for CFTR in which S341 was mutated to alanine (S341A); the affinity of DPC for the mutant channel was reduced by approximately 5-fold compared with wild type (KD = 1251 PM). Login to comment 53 ABCC7 p.Ser341Ala Figure4B shows thatthevoltagedependenceoftheweaker bindingfor S341A was changed only slightly from the wild-type. Login to comment 54 ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala In the S341A mutation, Cl-permeation in the absence of blockers was also changed: the S341A whole-cell currents rectified inwardly (Figure 3C), opposite to wild-type currents. Login to comment 58 ABCC7 p.Ser341Thr 625 Tnplc This importance of S341 is supported by data on S341T, in which a hydroxyl group is conserved on the side chain. Login to comment 59 ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala ABCC7 p.Ser341Thr The S341T mutation gives a Kn for DPC binding intermediate to those of S341A and the wild type (Table 1) and a smaller degree of inward rectification than that of S341A (data not shown). Login to comment 60 ABCC7 p.Ser341Thr We believe that DPC interacts with the -OH of S341, probably through hydrogen bonding of the carboxyl moiety, but that DPC has limited flexibility in its binding configuration with the wild-type channel, since S341T did not fully restore the affinity for DPC to wild-type levels. Login to comment 61 ABCC7 p.Ser1141Ala The DPC-Binding Site Can Be Moved to TM 12 S1141 in TM 12 had a predicted position analogous to that of S341 in TM 6 (Figure I), yet mutation S1141A I (/AA) Figure 3. Login to comment 68 ABCC7 p.Ser341Ala (0 S341A mutant, showing strong inward rectification and decreased DPC block. Login to comment 69 ABCC7 p.Ser341Ala (D) Triple mutation (S341A-M11401-Tll- 42F), showing strong inward rectification and restored DPC block compared with (C). Login to comment 70 ABCC7 p.Thr1134Phe (E) T1134F mutation, showing increased block at all voltages. Login to comment 71 ABCC7 p.Lys335Glu (F) K335E mutation, indicating inward rectification and unchanged DPC binding affinity. Login to comment 73 ABCC7 p.Ser341Ala S1141 could, however, participate in a binding site for DPC when the binding site including S341 was removed (S341A) and when the methionineand threonine residues immediatelyadja- cent to S1141 were changed to match the isoleucine and phenylalanine residues immediately adjacent to S341. Login to comment 74 ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala ABCC7 p.Thr1142Phe That is, the triple mutant S341A-M11401-T1142F bound DPC more tightly than S341A alone, with an affinity close to that of the wild-type channel (Figure 3D; Figure 4). Login to comment 78 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser341Thr ABCC7 p.Thr339Ala Affinity and Voltage Dependence for Block of CFTR Variants by DPC Construct TM Ko( - 100) (PM) 0 I-V Relation n Properties Wild type Wild type low [Cl-], (10 mM) K335E 6 K335F 6 T338A 6 T339A 6 S341A 6 S341T 6 S1118A 11 T1134A 12 T1134F 12 S1141A 12 Triple 6,12 276 f 14 181 f 13" 303 -t 14 351 * 15' 220 * 14 284 * 47 1251 f 116a 530 f 80" 243 * 37 230 * 20 74 * 3" 220 * 13 325 * 26b 0.41 f 0.01 0.32 f 0.02" 0.42 f 0.01 0.42 f 0.02 0.36 f 0.02" 0.44 * 0.12 0.49 * 0.03" 0.35 f 0.09 0.40 f 0.02 0.35 * 0.02" 0.41 f 0.01 0.42 f 0.03 0.21 * O.Ol",b Linear, E,,, = -8 f 1 mV Ere\ = +48+2mV Inward rectification Linear Linear Linear Strong inward rectification Inward rectification Linear Linear Linear Linear Strong inward rectification Affinity for DPC was determined empirically at -100 mV, from whole-cell currents measured in the presence of 200 uM DPC (see Experimental Procedures). Login to comment 84 ABCC7 p.Ser341Ala ABCC7 p.Thr1142Phe E,,,, the reversal potential, determined empirically from the voltage steps; Triple, S341A-M11401-T1142F mutant. Login to comment 86 ABCC7 p.Ser341Ala b p < .025 by unpaired t test compared with mutant S341A. Login to comment 91 ABCC7 p.Lys335Phe In TM 6, K335F displayed no change in rectification or 0 but slightly weakened DPC binding (Table 1). Login to comment 92 ABCC7 p.Lys335Glu In contrast, K335E displayed slight inward rectification of unblocked currents, but this mutant had the same DPC affinity as wild-type CFTR (Figure 3F). Login to comment 93 ABCC7 p.Thr1134Phe In TM 12, the T1134F mutation increased DPC affinity to 74 f 3 PM but did not change 8 (Figure 3E; Figure 4), suggesting that this mutation stabilizes DPC binding at its main site, S341. Login to comment 95 ABCC7 p.Thr1134Phe The increased binding of DPC on the T1134F mutant was confirmed at the single-channel level, as described below. Login to comment 96 ABCC7 p.Thr1134Phe T1134F single channels recorded in excised, inside-out patches had a smaller amplitude than wild type, with conductance y = 5.8 f 0.2 pS (range 4.1-6.4 pS; n = 14; Figure 5) versus y = 8.0 + 0.4 pS for wild type (McCarty et al., 1993). Login to comment 98 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe As with S341A, however, the T1134F mutant channel kinetics were qualitatively similar to wild type, with seconds-long openings, uninterrupted at positive voltages and interrupted by brief closures at negative voltages (Figure 5; Figure 7). Login to comment 99 ABCC7 p.Thr1134Phe Also like the wild type, T1134F occasionally showed a long-lived subconductance state, with amplitude -60% of the full conductance. Login to comment 100 ABCC7 p.Thr1134Phe As noted previously for the wild-type channel (McCarty et al., 1993), the subconductance state in T1134F was not blocked by DPC (Figure 6B), as though DPC cannot reach its binding site at S341 when the channel is in this state. Login to comment 101 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe A Further Test for Open-Channel Block Application of DPC to the cytoplasmic surface of excised inside-out patches resulted in blocker-induced closures of 3-to Qfold longer duration for T1134F than for wild type, consistent with the increased DPC affinity of the T1134F mutation (Figure 6). Login to comment 102 ABCC7 p.Thr1134Phe The longer residence time of DPC on the T1134F channel enabled us to resolve the kinetics of blockade at several DPC concentrations with V, = -100 mV. Login to comment 104 ABCC7 p.Thr1134Phe The DPC-induced closed times of T1134F averaged 1.8 + 0.1 ms (averaged from 20, 50, and 100 PM) and were independent of DPC concentration (Table 2; Figure 7; Figure 8). Login to comment 111 ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Thr1142Phe Open circles, wild-type; closed circles, T1134F; open boxes, S341A; closed boxes, triple mutation (S341A-Mll- 401-T1142F). Login to comment 127 ABCC7 p.Thr1134Phe Exceptions were the blockade of outward currents for T1134F, owing to the greatly increased affinity for DPC, and the triple mutation, owing to the changed voltage dependence of block. Login to comment 137 ABCC7 p.Thr1134Phe For the T1134F mutation, which has kinetics favorable for detailed study, the rate constant for blocking (inverse of the open time constant) increases linearly with [DPC], but the rate constant for unblocking is independent of [DPC]. Login to comment 138 ABCC7 p.Thr1134Phe 620 A Wild-type T1134F (TM-1 2) S341 A (TM-6) Figure 5. Login to comment 139 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe Mutations 5341A and T1134F Lower Single-Channel Conductance without Changing Kinetics Wild-type and T1134F traces are from inside-out patches excised in 1 mM ATP. Login to comment 143 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe (A) Comparison of wild-type, T1134F, and S341A conductances, all with V,, = -100 mV and F, flow-pass cutoff frequency) = 1 kHz. Login to comment 144 ABCC7 p.Ser341Ala (B) S341A single-channel traces with expanded amplitudescale,filteredat F, = 100 Hz to resolve openings clearly. Login to comment 146 ABCC7 p.Thr1134Phe The same is true for wild-type (McCarty et al., 1993) and T1134F (data not shown) channels. Login to comment 151 ABCC7 p.Thr1134Phe Although many observations presented here are consistent with the simple view that DPC is an open-channel blocker at wild-type and mutant CFTR channels, a complication is added by the observations that unblocked T1134F displays an extra closed time constant compared with wild type, and that this time constant is similar to the residence time of DPC. Login to comment 153 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe We believe the unblocked T1134F channel only coincidentally has a time constant near that of the lifetime of DPC on the T1134F channel. Login to comment 155 ABCC7 p.Ser341Ala The Pore of CFTR Is lined by TM 6 and TM 12 We conclude that TM 6 lines the pore because mutation S341A lowers the single-channel conductance by a factor of 8, reverses the direction of rectification, and removes most binding of the open-channel blocker DPC. Login to comment 156 ABCC7 p.Lys335Glu ABCC7 p.Lys335Phe Also in TM 6, mutation K335F slightly reduces DPC block, and mutation K335E gives inward rectification (see also Tabcharani et al., 1993). Login to comment 157 ABCC7 p.Thr1134Phe In TM 12, mutation T1134F strongly increases DPC affinity, as confirmed by single-channel measurements, and significantly lowers the single-channel conductance. Login to comment 158 ABCC7 p.Ser341Ala ABCC7 p.Thr1142Phe The sequence M11401-S1141-T1142F in TM 12 restores DPC binding to the S341A mutant. Login to comment 160 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe The identification of 629 B - * C T1134F T1134F Unblocked 50 pM DPC 810 / 0123456789 0 1 2 3 4 5 6 7 8 9 (msec) D Time Figure 6. Login to comment 161 ABCC7 p.Thr1134Phe The Residence Time of DPC Is Longer on the T1134F Channel than on the Wild-Type Channel Single-channel records are from inside-out patches excised in 1 mM ATP with V, = -100 mV, filtered at 1 kHz. Login to comment 168 ABCC7 p.Thr1134Phe (C) Representative closed time histogram for the unblocked T1134F channel. Login to comment 169 ABCC7 p.Thr1134Phe (D) Representative closed time histogram for T1134F with 50 uM DPC added to the cytoplasmic side. Login to comment 173 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Lys335Glu Previous studies of CFTR have found that mutations of positively charged residues in TM 1 and TM 6 (including K335E) give small changes in ionic selectivity (Anderson et al., 1991b) and that naturally occurring mutations of positively charged residues just extracellular to TM 2, or within TM 6 (R347P and R334W), reduce single-channel conductance and alter kinetics (Sheppard et al., 1993). Login to comment 178 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Ser341Ala Mutations R334W, R347P, and S341A may reduce single-channel conductance by removing a Cl--binding site. Login to comment 200 ABCC7 p.Thr1134Phe Time Constants for Block of T1134F Single Channels by Various DPC Concentrations IDPCI n Area, T,? Login to comment 208 ABCC7 p.Thr1134Phe Further Kinetic Analysis of the Data for DPC Blockade of T1134F Open circles, I/T, where T represents open time; closed circles, l/r, where T represents the second (DPC-induced) closed time constant within a burst. Login to comment 219 ABCC7 p.Ser341Ala The mutation that produces the largest effect on DPC block, S341A, also lowers the single-channel conductance from 8 to 1 pS and changes the rectification from outward to inward. Login to comment 223 ABCC7 p.Ser341Ala The possibility that the effects of S341A are allosteric is also rendered less likely by the intermediate effect on both drug block and rectification of replacing S341 with another hydroxylated residue, threonine. Login to comment 225 ABCC7 p.Ser341Thr The conservative S341T mutation alone weakens DPC block enough to implicate residue S341 in binding DPC. Login to comment 226 ABCC7 p.Thr338Ala ABCC7 p.Thr339Ala In addition, other mutations of hydroxylated residues in TM 6, namely T338A and T339A, affect neither DPC block nor rectification (Table 1). Login to comment 227 ABCC7 p.Ser341Ala This argues against the proposal that any alteration in sequencewithinTM6causes nonspecific effectsand isfurther evidenceforthespecificityof the S341A mutation. Login to comment 228 ABCC7 p.Thr1134Phe Similarly, in TM 12, mutation T1134F strengthens DPC binding and reduces single-channel conductance from 8 to <6 pS. Login to comment 229 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe DPC binds at the same electrical position, 40% of the distance through the membrane field, in the T1134F mutant as in the wild type, evidence that T1134F makes no gross disruption in the binding site. Login to comment 230 ABCC7 p.Thr1134Ala Control mutation T1134A has insignificant effects on DPC binding, as does mutation SIIIBA, in TM 11 at a position homologous to T1134. Login to comment 240 ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe The relative orientation of the side chains on the phenylalanines of K335F and T1134F may explain the opposite results of phenylalanines placed at these nearly equivalent positions. Login to comment 241 ABCC7 p.Thr1134Phe In our model of TM 12, the phenyl ring of T1134F points directly into the pore and lies along and perpendicular to the second phenyl NeUrClfl 632 Figure 9. Login to comment 252 ABCC7 p.Lys335Phe In TM 6, the K335F mutation slightly decreased DPC binding affinity. Login to comment 267 ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe MDR has a rather broad substrate specificity, perhaps be- (C) Same as (B), with mutations K335F and T1134F highlighted. Login to comment 268 ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe Note that K335F lies parallel to the second phenyl ring of DPC, and T1134F lies perpendicular. Login to comment 299 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe Single-channel currents were recorded from manually stripped oocytes injected with 70 ng of T1134F cRNA or with 100 ng of S341A plus 0.8 ng of &adrenergic receptor cRNA. Login to comment 308 ABCC7 p.Thr1134Phe T1134F single-channel traces were amplified at 20 dB per decade during acquisition. Login to comment 309 ABCC7 p.Ser341Ala For the S341A mutation, which results in singlechan- nels of very low conductance, currents were filtered at 100 Hz and amplified at 20 dB per decade during acquisition. Login to comment