ABCMdb

PMID: 18596043

Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB1 p.Gly251Val To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). Login to comment 7 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Login to comment 21 ABCB1 p.Leu65Arg ABCB1 p.Gly251Val It was found that the L65R mutation promoted maturation of P-gp processing mutants such as ⌬Y490 and G251V (9). Login to comment 37 ABCB1 p.Leu65Arg Printed in the U.S.A. with a L65R substitution was previously found to be particularly efficient (9). Login to comment 45 ABCB1 p.Gly251Val Accordingly,wefirstperformedarginine-scanningmutagenesis on TM1 of a P-gp processing mutant (G251V) to test their effects on maturation of the mutant. Login to comment 48 ABCB1 p.Met68Arg The most efficient rescue was observed with the M68R mutant. Login to comment 53 ABCB1 p.Gly251Val Mutations were introduced into wild-type or processing mutant G251V (13) P-gp cDNAs as described previously (6, 15). Login to comment 54 ABCB1 p.Gly251Val To perform arginine-scanning mutagenesis of TM1, the cDNA of mutant G251V P-gp was modified to create a set of mutants that contained one arginine at positions 52-72. Login to comment 55 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg ABCB1 p.Val71Arg For disulfide cross-linking analysis, the cDNA of mutant L339C(TM6)/F728C(TM7) (16) was modified to also encode the G64R, M68R, or V71R mutations. Login to comment 71 ABCB1 p.Gly251Val RESULTS Effect of TM1 Arginines on Maturation of Processing Mutant G251V-The crystal structure of the bacterial ABC drug transporter Sav1866 showed that the 12 TM segments of the transport complex surrounded a funnel-shaped chamber exposed to the extracellular environment (24). Login to comment 81 ABCB1 p.Leu65Arg We previously observed that introduction of arginines into TM segments such as L65R (TM1), T199R(TM3), I306R(TM5), and F343R(TM6) promoted maturation of P-gp processing mutants (9). Login to comment 91 ABCB1 p.Gly251Val Therefore, processing mutant G251V was selected for analysis as it shows inefficient maturation relative to wild-type P-gp (33). Login to comment 92 ABCB1 p.Gly251Val The G251V mutation is located in the first intracellular loop (see Fig. 1A). Login to comment 97 ABCB1 p.Gly251Val Immunoblot analysis of the G251V processing mutant, however, showed that the major product was the 150-kDa immature protein along with a minor amount (about 20%) of mature 170-kDa product (Fig. 2). Login to comment 98 ABCB1 p.Gly251Val The most common effect of introducing arginines into mutant G251V was to reduce the level of mature P-gp (12 of 21 mutants). Login to comment 100 ABCB1 p.Gly251Val Fig. 2 shows that the presence of an arginine at these positions reduced the level of mature product in mutant G251V from about 20% to less than 5% (Fig. 2B). Login to comment 101 ABCB1 p.Gly251Val Introduction of arginines at positions 52 or 61 had little effect on maturation of G251V P-gp. Login to comment 102 ABCB1 p.Gly251Val Introduction of arginines at the extracellular half of TM1 (positions 64, 65, 68, and 71), however, increased maturation of mutant G251V. Login to comment 103 ABCB1 p.Leu65Arg ABCB1 p.Met68Arg Mutants M68R and L65R showed the highest increase in maturation efficiency (85 and 60% mature 170-kDa mature, respectively) (Fig. 2). Login to comment 104 ABCB1 p.Gly64Arg ABCB1 p.Val71Arg Mutants G64R and V71R showed maturation efficiencies of FIGURE 1. Login to comment 109 ABCB1 p.Leu65Arg The position of the L65R mutation that promotes maturation of P-gp processing mutants is shown as a black ball. Login to comment 116 ABCB1 p.Leu65Arg The position of Leu-65 (L65R mutation promotes maturation of processing mutants) is shaded in black. about 45%. Login to comment 117 ABCB1 p.Met69Arg ABCB1 p.Leu67Arg ABCB1 p.Pro66Arg ABCB1 p.Leu70Arg ABCB1 p.Phe72Arg Introduction of arginines at other positions at the extracellular end of TM1 had little effect (10-20% mature P-gp in mutants L67R, M69R, and L70R) or reduced the level of mature product to less than 10% of total P-gp (mutants P66R and F72R). Login to comment 118 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg ABCB1 p.Val53Arg Mutants G251V, V53R/G251V (inhibited maturation) and M68R/G251V (enhanced maturation) were subjected endoglycosidase H or F digestion to determine whether the alterations in mobility of the mutant P-gps were indeed due to changes in the glycosylation state of the protein. Login to comment 125 ABCB1 p.Gly251Val ABCB1 p.Met68Arg Because the M68R mutation was most efficient in promoting maturation of the G251V processing mutant, we tested whether it could also promote maturation of a different processing mutant. Login to comment 126 ABCB1 p.Met68Arg Accordingly, the M68R mutation was introduced into the ⌬NBD2- P-gp processing mutant (20). Login to comment 128 ABCB1 p.Met68Arg ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala Mutants ⌬NBD2- P-gp, M68R/⌬NBD2-P-gp, and ⌬NBD2-P-gp lacking the three glycosylation sites (N91A, N94A, N99A) were expressed in HEK 293 cells, and whole cell SDS extracts were subjected to immunoblot analysis. Login to comment 129 ABCB1 p.Met68Arg Fig. 4 shows that the M68R mutant yielded an extra P-gp protein that corresponded in size to the mature protein (Fig. 4). Login to comment 130 ABCB1 p.Met68Arg Treatment with endoglycosidases H and F showed that the immature form (100 kDa) was sensitive to endoglycosidases H and F, whereas the slower migrating M68R protein (115 kDa) was sensitive to endoglycosidase F but not endoglycosidase H (data not shown). Login to comment 131 ABCB1 p.Gly251Val ABCB1 p.Gly251Val Drug Rescue of P-gp G251V Arginine Mutants That Do Not Show Enhanced Maturation-If the arginine mutants that showed reduced maturation relative to the G251V (such as FIGURE 2. Login to comment 132 ABCB1 p.Gly251Val Effect of TM1 arginine mutations on maturation of the G251V processing mutant. Login to comment 133 ABCB1 p.Gly251Val A, immunoblot analysis of whole cell extracts of HEK 293 cells expressing no P-gp (Control), wild-type P-gp (Wild-type), or a G251V processing mutant containing the indicated TM1 arginine mutations. Login to comment 138 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg ABCB1 p.Val53Arg Whole cell SDS extracts of HEK 293 cells expressing A52-tagged mutants G251V, V53R/G251V, or M68R/G251V were treated with endoglycosidase Hf (H),peptideN-glycosidaseF(F)ornoendoglycosidase(-).Equivalentamountsweresubjectedtoimmunoblot analysis. Login to comment 140 ABCB1 p.Val53Arg V53R, Fig. 2A) were not grossly misfolded, then it should be possible to promote their maturation by carrying out expression in the presence of drug substrates. Login to comment 142 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met69Arg ABCB1 p.Thr55Arg Therefore, mutants showing reduced (T55R/G251V) or similar maturation efficiencies (H61R/G251V, M69R/G251V) to the G251V parent were expressed in the presence of 5 ␮M cyclosporin A, 30 ␮M verapamil, 5 ␮M vinblastine, or 30 ␮M rhodamine B. Login to comment 143 ABCB1 p.Gly251Val These drug concentrations promoted rescue of the G251V parent so that mature (170 kDa) protein was the major product in the treated cells (Fig. 5). Login to comment 144 ABCB1 p.Gly251Val ABCB1 p.Thr55Arg By contrast, a mutant where the introduced arginine inhibited maturation of G251V (mutant T55R) could not be rescued with any of the drug substrates (Fig. 5). Login to comment 146 ABCB1 p.Gly251Val For example, only cyclosporin A rescued mutant H61R/G251V (Fig. 5). Login to comment 147 ABCB1 p.Gly251Val ABCB1 p.Met69Arg By contrast, all drugs except verapamil rescued mutant M69R/G251V (Fig. 5). Login to comment 148 ABCB1 p.Gly251Val The effects of drug substrates on the expression of the other arginine/G251V mutants are summarized in Table 1. Login to comment 149 ABCB1 p.Leu65Arg ABCB1 p.Met68Arg Mutants L65R and M68R yielded mature 170-kDa P-gp as the major product in the absence of drug substrates. Login to comment 150 ABCB1 p.Gly251Val ABCB1 p.Leu67Arg ABCB1 p.Leu70Arg ABCB1 p.Val52Arg Mutants V52R, L67R, and L70R resembled the parent (G251V) P-gp, as expression in any of the drug substrates promoted efficient maturation (mature P-gp became the major product) of the protein. Login to comment 151 ABCB1 p.Pro66Arg ABCB1 p.Phe72Arg ABCB1 p.Val53Arg ABCB1 p.Thr55Arg ABCB1 p.Ala57Arg ABCB1 p.Ala63Arg ABCB1 p.Gly62Arg ABCB1 p.Gly54Arg ABCB1 p.Ile59Arg ABCB1 p.Leu56Arg ABCB1 p.Ala58Arg ABCB1 p.Ile60Arg By contrast, 12 of the mutants (V53R, G54R, T55R, L56R, A57R, A58R, I59R, I60R, G62R, A63R, P66R, and F72R) showed little or no rescue in the presence of drug substrates. Login to comment 153 ABCB1 p.Gly64Arg Mutant G64R only showed significant rescue with cyclosporin A and rhodamine B. Login to comment 155 ABCB1 p.Gly251Val ABCB1 p.Met68Arg Effect of Suppressor Arginine Mutations on P-gp-Drug Interactions-Because mutant M68R/G251V showed efficient maturation (about 85%) when expressed in the absence of drug substrates (Fig. 2), we tested whether Arg-68 affected P-gp- drug interactions in disulfide protection assays using mutant L339C(TM6)/F728C(TM7) as described previously (9). Login to comment 156 ABCB1 p.Gly64Arg ABCB1 p.Gly251Val ABCB1 p.Val71Arg Because the G64R and V71R changes also promoted maturation of the G251V processing mutant (Fig. 2), we also introduced these mutations into the L339C(TM6)/F728C(TM7) mutant. Login to comment 159 ABCB1 p.Met68Arg Effect of the M68R mutation on maturation of the ⌬NBD2 processing mutant. Login to comment 160 ABCB1 p.Met68Arg ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala Whole cell extracts of HEK 293 cells expressing mutants ⌬NBD2-P-gp with no changes (None), the M68R mutation, or N91A, N94A and N99A changes to the glycosylation sites (Unglycos) were subjected to immunoblotanalysis.Thepositionsofmature,immature,andunglycosylated(Unglycos) forms of ⌬NBD2 P-gp are indicated. Login to comment 163 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met69Arg ABCB1 p.Thr55Arg HEK 293 cells expressing mutants G251V, T55R/G251V, H61R/G251V, or M69R/G251V were treated with no drug (None), 5 ␮M cyclosporin A (Cyclo), 30 ␮M verapamil (Ver), 5 ␮M vinblastine (Vin), or 30 ␮M rhodamine B (Rhod) for 24 h. Whole cell extracts were then subjected to immunoblot analysis. Login to comment 165 ABCB1 p.Gly64Arg ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg ABCB1 p.Val71Arg ABCB1 p.Met69Arg ABCB1 p.Pro66Arg ABCB1 p.Leu70Arg ABCB1 p.Phe72Arg ABCB1 p.Val53Arg ABCB1 p.Thr55Arg ABCB1 p.Val52Arg ABCB1 p.Ala57Arg ABCB1 p.Ala63Arg ABCB1 p.Ile59Arg ABCB1 p.Leu56Arg ABCB1 p.Ala58Arg ABCB1 p.Ile60Arg TABLE 1 Effects of drug substrates on the maturation of TM1 arginine mutants containing the G251V mutation Mutation (G251V ؉) No drug Cyclosporin A Verapamil Vinblastine Rhodamine None - 111a,c 111 111 11 V52R -a,b 111 111 111 11 V53R 2 - - - - G64R 2 - - - - T55R 2 - - - - L56R 2 - - - - A57R 2 - - - - A58R 2 - - - - I59R 2 - - - - I60R 2 - - - - H61R - 111 1 1 1 G62R 2 - - - - A63R 2 - - - - G64R 1 111 1 1 11 L65R 11 111 11 11 11 P66R 2 1 - - - L67R - 111 111 111 11 M68R 111 111 111 111 111 M69R - 111 - 11 111 L70R - 111 111 111 11 V71R 1 111 111 111 11 F72R 2 1 1 1 1 a Change in the amount of mature (170 kDa) protein in the presence of drug substrate relative to that in the absence of drug substrate. Login to comment 167 ABCB1 p.Gly251Val c Change in the amount of mature (170 kDa) protein relative to the mutant G251V expressed in the absence of drug substrate. Login to comment 172 ABCB1 p.Leu65Arg In a previous cross-linking study we found that introduction of L65R change into mutant L339C(TM6)/ F728C(TM7) reduced its apparent affinity for vinblastine by about 60-fold but had little effect on P-gp interactions with cyclosporin A or rhodamine B (9). Login to comment 173 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg ABCB1 p.Val71Arg Membranes were prepared from HEK 293 cells expressing mutants L339C(TM6)/F728C(TM7), G64R/ L339C(TM6)/F728C(TM7), M68R/ L339C(TM6)/F728C(TM7), and V71R/L339C(TM6)/F728C(TM7). Login to comment 177 ABCB1 p.Met68Arg A representative example of the effects of various concentrations of vinblastine on cross-linking of mutant M68R/L339C(TM6)/ F728C(TM7) by M14M is shown in Fig. 6. Login to comment 179 ABCB1 p.Met68Arg The M68R/ L339C(TM6)/F728C(TM7) mutant, however, required 266 ␮M vinblastine (Ͼ500-fold increase) to inhibit cross-linking by 50%. Login to comment 181 ABCB1 p.Gly64Arg ABCB1 p.Val71Arg The G64R/L339C(TM6)/ F728C(TM7) mutant also showed a large reduction in apparent affinity for vinblastine (Ͼ100-fold), whereas the V71R change had little effect. Login to comment 183 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Met68Arg The G64R, L65R, and M68R mutations preferentially affected P-gp-vinblastine interactions. Login to comment 185 ABCB1 p.Leu65Cys It was shown that mutant L65C could be labeled with a thiol-reactive derivative of verapamil to cause permanent activation of ATPase activity. Login to comment 188 ABCB1 p.Met68Arg Effect of the M68R mutation on inhibition of cross-linking by vinblastine. Login to comment 189 ABCB1 p.Met68Arg A, membranes were prepared from HEK 293 cells expressing mutants L339C(TM6)/F728C(TM7) or M68R/L339C(TM6)/F728C(TM7). Login to comment 194 ABCB1 p.Gly64Arg ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Phe728Cys ABCB1 p.Met68Arg ABCB1 p.Val71Arg TABLE 2 Concentrations of drug substrates required to inhibit cross-linking by 50% Mutant Vinblastine Cyclosporin A Rhodamine B ␮M ␮M ␮M L339C/F728C 0.5 Ϯ 0.2a 0.8 Ϯ 0.3 52 Ϯ 14 G64R/L339C/F728C 53 Ϯ 15b 1.5 Ϯ 0.4 61 Ϯ 10 M68R/L339C/F728C 266 Ϯ 62b 1.3 Ϯ 0.3 57 Ϯ 7 V71R/L339C/F728C 0.6 Ϯ 0.2 0.8 Ϯ 0.2 57 Ϯ 10 a Each value is the mean Ϯ S.D. (n ϭ 3-4 separate cross-linking experiments). Login to comment 197 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg Therefore, we measured drug stimulation of ATPase activity of mutants G64R and M68R using various concentrations of verapamil to test whether the apparent affinity for verapamil of the P-gp mutants was altered. Login to comment 200 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg Accordingly, mutations G64R or M68R were introduced into a wild-type P-gp that contained a histidine tag at the COOH-terminal end. Login to comment 201 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg Histidine-tagged wild-type and mutants G64R and M68R P-gps were expressed in HEK 293 cells, and the proteins were isolated by nickel-chelate chromatography. Login to comment 205 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg Verapamil also stimulated the ATPase activities of mutants G64R and M68R (1.6 and 1.8 ␮mol Pi/min/mg of protein, respectively). Login to comment 206 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg Although both mutants G64R and M68R showed relatively high activation of ATPase activity with verapamil, their apparent affinities for verapamil were reduced by about 19-and 4-fold, respectively. Login to comment 207 ABCB1 p.Gly64Arg ABCB1 p.Met68Arg Half-maximal activation of the ATPase activities of mutants G64R and M68R required 540 ␮m and 120 ␮M verapamil, respectively, compared with 29 ␮m for wild-type P-gp. Login to comment 208 ABCB1 p.Leu65Arg ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg Effect of TM11 Mutations on Rescue of Mutant G251V Containing the L65R and M68R Changes-One reason that Arg-68 caused the largest increase in maturation of mutant G251V is that the arginine residue may promote packing of the TM segments. Login to comment 214 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg ABCB1 p.Met68Arg To test if Tyr-950 or Tyr-953 influences the ability of the M68R mutation to promote maturation of the G251V mutant, we introduced the Y950A or Y953A mutations into mutant M68R/ G251V mutant. Login to comment 216 ABCB1 p.Gly251Val ABCB1 p.Met68Arg Fig. 8 shows the maturation efficiencies compared with the M68R/G251V parent. Login to comment 217 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala The presence of the Y950A or Y953A mutation reduced the maturation efficiency to less than 50% (Fig. 8A, lanes 6 and 9). Login to comment 218 ABCB1 p.Gly251Val ABCB1 p.Met68Arg Not all mutations in TM11, however, affected maturation of the M68R/G251V mutant. Login to comment 219 ABCB1 p.Gln946Ala ABCB1 p.Phe951Ala ABCB1 p.Ser952Ala ABCB1 p.Met949Ala ABCB1 p.Met948Ala ABCB1 p.Gly251Val ABCB1 p.Met68Arg The presence of mutations Q946A, M948A, M949A, F951A, or S952A did not appear to affect the maturation of the M68R/ G251V mutant (Fig. 8A, lanes 2-5, 7, and 8). Login to comment 220 ABCB1 p.Leu65Arg ABCB1 p.Leu65Arg ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val ABCB1 p.Gly251Val Because the L65R mutation also promoted maturation of mutant G251V, we tested whether the Y950A or Y953A mutations would have any effect on maturation of the L65R/G251V mutant. Login to comment 221 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala Fig. 8B shows that introduction of the Y950A mutation caused a relatively large reduction in the maturation efficiency of the mutant (Fig. 8B, lane 3), whereas the Y953A mutation had little effect (Fig. 8B, lane 4). Login to comment 222 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val ABCB1 p.Met68Arg In contrast, both Y950A and Y953A affected the maturation efficiency of the M68R/G251V mutant. Login to comment 223 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val The Y950A and Y953A mutations did not affect maturation of the (G251V) parent (Fig. 8A, lanes 13 and 14) or wild-type P-gp (data not shown). Login to comment 224 ABCB1 p.Gly251Val ABCB1 p.Gly251Val These results suggest that interactions between Arg-65(TM1) and Tyr-950(TM11), Arg-68(TM1) and Tyr-950(TM11), or Arg-68 (TM1) and Tyr953(TM11) may be responsible for the enhanced maturation of the G251V mutant. Login to comment 228 ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe Conservative replacement of Tyr-950 or Tyr-953 with Phe would only disrupt hydrogen bond interactions. Login to comment 229 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Tyr950Phe ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe ABCB1 p.Met68Arg ABCB1 p.Met68Arg Mutants L65R/G251V/Y950F, M68R/G251V/ Y950A/Y953A, and M68R/G251V/Y950F/Y953F were constructed, and the cDNAs were expressed in HEK 293 cells. Login to comment 230 ABCB1 p.Tyr953Ala ABCB1 p.Gly251Val ABCB1 p.Tyr950Phe Only the Y950F change was introduced into the L65R/G251V mutant since the Y953A change did not affect maturation of the mutant (Fig. 8B, lane 4). Login to comment 231 ABCB1 p.Tyr953Ala ABCB1 p.Tyr950Ala ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Tyr950Phe ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe ABCB1 p.Met68Arg Immunoblot analysis shows the Y950A/Y953A and Y950F/Y953F changes in M68R/G251V (Fig. 8A, lanes 11 and 12) or Y950F change in L65R/G251V (Fig. 8B, lane 5) reduced maturation to levels similar to that observed in the G251V parent. Login to comment 232 ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe Introduction of the Y950F and Y953F FIGURE 7. Login to comment 233 ABCB1 p.Met68Arg Verapamil-stimulated ATPase activities of wild-type and mutant G64R and M68R P-gps. Login to comment 237 ABCB1 p.Gly251Val changes into the G251V parent did not affect its maturation efficiency (Fig. 8, A, lane 15, and B, lane 6). Login to comment 239 ABCB1 p.Gly251Val DISCUSSION It was found that maturation of mutant G251V was not inhibited when arginines were introduced at many positions on the extracellular half of TM1. Login to comment 261 ABCB1 p.Gly251Val Cyclosporin A and rhodamine B but not vinblastine or verapamil promoted maturation of the mutant G64R/ G251V (Table 1). Login to comment 262 ABCB1 p.Gly251Val The H61R mutation appeared to perturb vinblastine interactions as expression in the presence of vinblastine did not promote maturation of the mutant H61R/G251V (Fig. 5). Login to comment 264 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg Effect of TM11 mutations on maturation of M68R/G251V and L65R/G251V mutants. Login to comment 265 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg A, HEK 293 cells were transfected with mutants M68R/G251V (lanes 2-12) or G251V (lanes 1, 13-15) containing the indicated TM11 mutations. Login to comment 267 ABCB1 p.Gly251Val ABCB1 p.Gly251Val B, HEK 293 cells were transfected with mutants M65R/G251V (lanes 2-5) or G251V (lanes 1, 6) containing the indicated TM11 mutations. Login to comment 270 ABCB1 p.Ala57Leu results are consistent with a previous study that examined the effects of arginine substitutions to residues Ala-57 to Leu-65 in TM1 on drug transport by P-gp (43). Login to comment 276 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Met68Arg ABCB1 p.Met69Arg Mutants H61R/G251V or M69R/G251V showed little drug rescue with verapamil (Fig. 5), and mutants G64R and M68R in a wild-type background showed reductions in apparent affinity for verapamil in ATPase assays (Fig. 7). Login to comment 278 ABCB1 p.Val71Arg Residue Val-71, however, appeared to lie outside the drug binding pocket since the V71R mutation did not affect P-gp-drug interactions (Table 3). Login to comment 280 ABCB1 p.Met68Arg The observation that the M68R mutation can reduce the apparent affinities for the drug substrates verapamil and vinblastine would be unexpected because it appears that P-gp initially interacts with drug substrates in the inner leaflet of the bilayer (44). Login to comment 281 ABCB1 p.Met68Arg One explanation is that the M68R mutation causes an indirect effect on the vinblastine and verapamil binding sites. Login to comment 282 ABCB1 p.Met68Arg The structural alterations may be small because the M68R change did not appear to affect P-gp interactions with cyclosporin A or rhodamine B (Table 2). Login to comment 283 ABCB1 p.Met68Arg Another explanation is that the M68R mutation has altered the predicted "OFF-sites" of P-gp. Login to comment 285 ABCB1 p.Met68Arg Because the OFF-sites would be involved in drug release, they may be located in the outer portions of P-gp and would be sensitive to mutations such as M68R. Login to comment 291 ABCB1 p.Gly251Val Shading on the P-gp amino acids represents the effects of arginine substitutions on maturation of mutant G251V; the white, gray, and black-filled circles represent inhibition, no effect, or promotion of maturation, respectively. Login to comment 298 ABCB1 p.Gly251Val ABCB1 p.Met68Arg ABCB1 p.Val71Arg Some mutations such as G64R, L65R, M68R, and V71R actually promoted maturation of the G251V mutant. Login to comment 299 ABCB1 p.Gly251Val ABCB1 p.Met68Arg The L65R and M68R mutations were particularly effective in promoting maturation as they increased maturation efficiency of the G251V mutant from about 20 to 60-85%. Login to comment 302 ABCB1 p.Gly251Val Processing mutations such as G251V may alter the orientation of one or more TM segments in the membrane to interfere with subsequent packing of the TM segments at the interface between TMD1 and TMD2. Login to comment 304 ABCB1 p.Gly251Val ABCB1 p.Met68Arg An alternative possibility to explain why the L65R and M68R mutations were particularly effective in promoting maturation of the G251V mutant is that they promoted packing of the TM segments by forming hydrogen bonds with Tyr-950 and/or Tyr-953 located in TM11. Login to comment 306 ABCB1 p.Gly251Val Processing mutations such as G251V appear to act as thermodynamic hurdles to inhibit packing of the TM segments between TMD1 and TMD2 (6). Login to comment 308 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe ABCB1 p.Met68Arg This possibility is supported by the observations that the conservative Y950F and Y953F mutations reduced maturation of the M68R/G251V mutant to levels observed with the G251V parent. Login to comment 309 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Tyr950Phe ABCB1 p.Tyr953Phe An arginine introduced at position 65 in TM1 only appeared to form a hydrogen bond with Tyr-950 as the Y950F mutation but not the Y953F change reduced maturation of the L65R/G251V mutant to levels observed with the G251V parent. Login to comment 312 ABCC7 p.Val232Asp For example, it was demonstrated that the cystic fibrosis-phenotypic mutation V232D in CFTR TM segment 4 likely inhibits folding of CFTR by forming an unnatural hydrogen bond with Gln-207 in TM3 (47). Login to comment 314 ABCB1 p.Met68Arg In P-gp, the introduction of the L65R or M68R mutations appears to promote folding by enhancing TM1-TM11 interactions through hydrogen bond formation with the Tyr-950 and/or Tyr-953 in TM11. Login to comment