ABCMdb

PMID: 23766510

Cheepala SB, Bao J, Nachagari D, Sun D, Wang Y, Zhong TP, Naren AP, Zheng J, Schuetz JD
Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.
J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC4 p.Thr796Met We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. Login to comment 35 ABCC4 p.Thr796Met Our studies demonstrate that the analogous mutation in human ABCC4 (T796M) also reduces protein expression. Login to comment 37 ABCC4 p.Thr796Met Our studies demonstrate that the analogous mutation in human ABCC4 (T796M) also reduces protein expression. Login to comment 45 ABCC4 p.Thr796Met In Vitro Translation-In vitro translation reactions were performed with the following expression plasmids: pcDNA3.1 ZF Abcc4, Abcc4 T804M, ABCC4, ABCC4 T796M, and pLUC (luciferase vector) using the transcription- and translation-coupled (TNT) kit (Promega Inc., Madison, WI) in the presence of [35 S]Met. Login to comment 47 ABCC4 p.Thr796Met In Vitro Translation-In vitro translation reactions were performed with the following expression plasmids: pcDNA3.1 ZF Abcc4, Abcc4 T804M, ABCC4, ABCC4 T796M, and pLUC (luciferase vector) using the transcription-and translation-coupled (TNT) kit (Promega Inc., Madison, WI) in the presence of [35 S]Met. Login to comment 49 ABCC4 p.Thr796Met Cell Culture-NIH3T3, HEK293, or the cells transiently expressing one of the following proteins, ZF Abcc4, ZF Abcc4 T804M, ABCC4, and ABCC4 T796M, were maintained in Dulbecco`s DMEM containing 4500 mg/liter glucose, 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, and 100 òe;g/ml streptomycin in a humidified 5% CO2 incubator at 37 &#b0;C. Login to comment 50 ABCC4 p.Thr796Met Where specified, NIH3T3 cells transiently expressing either pACGFP ABCC4 or T796M were incubated with 50 òe;g/ml cycloheximide for different time points. Login to comment 51 ABCC4 p.Thr796Met Cell Culture-NIH3T3, HEK293, or the cells transiently expressing one of the following proteins, ZF Abcc4, ZF Abcc4 T804M, ABCC4, and ABCC4 T796M, were maintained in Dulbecco`s DMEM containing 4500 mg/liter glucose, 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, and 100 òe;g/ml streptomycin in a humidified 5% CO2 incubator at 37 &#b0;C. Login to comment 52 ABCC4 p.Thr796Met Where specified, NIH3T3 cells transiently expressing either pACGFP ABCC4 or T796M were incubated with 50 òe;g/ml cycloheximide for different time points. Login to comment 65 ABCC4 p.Thr796Met Confocal Microscopy-NIH3T3 or HEK293 cells were seeded on coverslips and chamber slides 24 h before transfection with the plasmid DNAs (250 ng) encoding pAcGFP ZF Abcc4, ZF Abcc4 T804M, ABCC4, ABCC4 T796M, and pCAL plasmid encoding RFP-tagged endoplasmic reticulum resident protein calreticulin encoding plasmid (pCAL (Origene) by using Lipofectamine LTX Plus reagent (Invitrogen))according to the manufacturer`s protocols. Login to comment 67 ABCC4 p.Thr796Met Confocal Microscopy-NIH3T3 or HEK293 cells were seeded on coverslips and chamber slides 24 h before transfection with the plasmid DNAs (250 ng) encoding pAcGFP ZF Abcc4, ZF Abcc4 T804M, ABCC4, ABCC4 T796M, and pCAL plasmid encoding RFP-tagged endoplasmic reticulum resident protein calreticulin encoding plasmid (pCAL (Origene) by using Lipofectamine LTX Plus reagent (Invitrogen))according to the manufacturer`s protocols. Login to comment 71 ABCC4 p.Thr796Met Briefly, NIH3T3 cells transiently expressing either pAcGFP human ABCC4 or T796M were rinsed with ice-cold PBS and incubated with 10 mM EZ-Link sulfo-NHS-SS-biotin solution in PBS (pH 8.0) (Pierce) for 30 min at room temperature. Login to comment 72 ABCC4 p.Thr796Met Briefly, NIH3T3 cells transiently expressing either pAcGFP human ABCC4 or T796M were rinsed with ice-cold PBS and incubated with 10 mM EZ-Link sulfo-NHS-SS-biotin solution in PBS (pH 8.0) (Pierce) for 30 min at room temperature. Login to comment 102 ABCC4 p.Thr796Met H, loss of intermolecular interactions of T796M human ABCC4. Login to comment 103 ABCC4 p.Thr796Met H, loss of intermolecular interactions of T796M human ABCC4. Login to comment 121 ABCC4 p.Thr796Met Because the Thr at position 804 in ZF Abcc4 is conserved among Abcc4 subfamily members, we developed expression vectors encoding full-length human WT AcGFP-ABCC4 and produced an analogous mutation to T804M, T796M in PAcGFP ABCC4. Login to comment 122 ABCC4 p.Thr796Met Because the Thr at position 804 in ZF Abcc4 is conserved among Abcc4 subfamily members, we developed expression vectors encoding full-length human WT AcGFP-ABCC4 and produced an analogous mutation to T804M, T796M in PAcGFP ABCC4. Login to comment 123 ABCC4 p.Thr796Met ABCC4 T796M exhibited a strong reduction in the core-glycosylated mature band c (Fig. 2F), which was confirmed by treating WT ABCC4-expressing lysates with the N-glycanase, PNGase F (Fig. 2G). Login to comment 124 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 T796M exhibited a strong reduction in the core-glycosylated mature band c (Fig. 2F), which was confirmed by treating WT ABCC4-expressing lysates with the N-glycanase, PNGase F (Fig. 2G). Login to comment 125 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met Notably, WT human ABCC4 and T796M produced almost identical amounts of protein (Fig. 2H) when in vitro translated, indicating that, like ZF T804M, the T796M substitution in human ABCC4 has no effect on mRNA translation. To determine T796M ABCC4 subcellular localization, live cell confocal microscopy was performed. Login to comment 126 ABCC4 p.Thr796Met Cells were transiently transfected with either ABCC4 WT or T796M expression plasmids along with an expression plasmid for calreticulin, an ER resident protein. Login to comment 128 ABCC4 p.Thr796Met In contrast, a majority (b0e;95%) of ABCC4 T796M localized with the Golgi/ER marker, calreticulin, or more diffusely in the cytoplasm (Fig. 2, I and J). Login to comment 129 ABCC4 p.Thr796Met ABCC4 p.Thr796Met In contrast, a majority (&#2b0e;95%) of ABCC4 T796M localized with the Golgi/ER marker, calreticulin, or more diffusely in the cytoplasm (Fig. 2, I and J). Login to comment 130 ABCC4 p.Thr796Met ABCC4 p.Thr796Met We next determined if the T796M substitution in CL3 altered the stability of human ABCC4. Login to comment 131 ABCC4 p.Thr796Met NIH3T3 cells were transiently transfected with either WT ABCC4 or ABCC4 harboring T796M substitution. Login to comment 133 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met In contrast, ABCC4 T796M does not have a readily detectable mature ABCC4 (band c); however, the turnover of the immature ABCC4 (band b) for WT and T796M are almost identical, suggesting the immature form of ABCC4 and T796M ABCC4 has the same degradation rate. Login to comment 134 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met In contrast, ABCC4 T796M does not have a readily detectable mature ABCC4 (band c); however, the turnover of the immature ABCC4 (band b) for WT and T796M are almost identical, suggesting the immature form of ABCC4 and T796M ABCC4 has the same degradation rate. Login to comment 135 ABCC4 p.Thr796Met Cytoplasmic Loop 3 Helicity Is Altered and Associated with Temperature-sensitive Rescue-We hypothesized that the T796M mutation in the human ABCC4 affects the helical conformation of the CL3 region. Login to comment 136 ABCC4 p.Thr796Met The helix containing T796M substitution was iteratively interrogated 16 times, and the structures were determined at temperatures ranging from 270 to 602 K. Login to comment 137 ABCC4 p.Thr796Met The helix containing T796M substitution was iteratively interrogated 16 times, and the structures were determined at temperatures ranging from 270 to 602 K. Login to comment 147 ABCC4 p.Thr796Met F, immunoblots of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 WT and ABCC4 T796M mutant. Login to comment 148 ABCC4 p.Thr796Met F, immunoblots of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 WT and ABCC4 T796M mutant. Login to comment 151 ABCC4 p.Thr796Met J, quantification of HEK293 transfected cells with GFP-tagged human ABCC4 WT, ABCC T796M mutant, and RFP-tagged ER marker (calreticulin). Login to comment 152 ABCC4 p.Thr796Met J, quantification of HEK293 transfected cells with GFP-tagged human ABCC4 WT, ABCC T796M mutant, and RFP-tagged ER marker (calreticulin). Login to comment 153 ABCC4 p.Thr796Met Signals from the three channels were acquired independently, and the merged images are presented. Co-localization of ABCC4 WT and PM is indicated by a reddish blue, and co-localization of GFP ABCC4 T796M and RFP-ER is indicated by a yellow color. Login to comment 154 ABCC4 p.Thr796Met ABCC4 p.Thr796Met Signals from the three channels were acquired independently, and the merged images are presented. Co-localization of ABCC4 WT and PM is indicated by a reddish blue, and co-localization of GFP ABCC4 T796M and RFP-ER is indicated by a yellow color. Login to comment 155 ABCC4 p.Thr796Met K, NIH3T3 cells were transfected with ABCC4 WT, T796M, and cycloheximide (final concentration, 50 òe;g/ml) was added 24 h post-transfection. Login to comment 158 ABCC4 p.Thr796Met T796M mutant, indicating that the stability of Met-796 containing helix is worse than the wild type. Login to comment 159 ABCC4 p.Thr796Met T796M mutant, indicating that the stability of Met-796 containing helix is worse than the wild type. Login to comment 160 ABCC4 p.Thr796Met Based on the MD analysis, we hypothesized that expression of the ABCC4 T796M might be restored by reducing temperature to 28 &#b0;C. Login to comment 161 ABCC4 p.Thr796Met ABCC4 p.Thr796Met Based on the MD analysis, we hypothesized that expression of the ABCC4 T796M might be restored by reducing temperature to 28 &#b0;C. Login to comment 162 ABCC4 p.Thr796Met ABCC4 p.Thr796Met We next investigated if subphysiological temperatures rescued expression and plasma membrane localization of ABCC4 T796M. Login to comment 163 ABCC4 p.Thr796Met NIH3T3 cells were transiently transfected with either ABCC4 or T796M expression vectors; 24 h post-transfection, one set of cells was transferred to 28 &#b0;C incubation, and the other remained at 37 &#b0;C. Login to comment 165 ABCC4 p.Thr796Met ABCC4 p.Thr796Met For the total protein, T796M is reduced by 50% compared with ABCC4 at 37 &#b0;C, with the unexpected increase in the amount of the mature form ABCC4 T796M likely due to the additional 24-h incubation. Login to comment 166 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met For the total protein, T796M is reduced by 50% compared with ABCC4 at 37 &#b0;C, with the unexpected increase in the amount of the mature form ABCC4 T796M likely due to the additional 24-h incubation. Login to comment 167 ABCC4 p.Thr796Met ABCC4 p.Thr796Met Interestingly, the immature band b in T796M was dramatically reduced by this additional incubation period. Login to comment 168 ABCC4 p.Thr796Met ABCC4 p.Thr796Met However, at 28 &#b0;C, T796M expression and surface expression are strongly increased (Fig. 3, B and C). Login to comment 169 ABCC4 p.Thr796Met This result indicates that subphysiological temperatures can rescue T796M expression and restore plasma membrane localization. Login to comment 170 ABCC4 p.Thr796Met Human ABCC4 T796M peptide forms an unstable helix. Login to comment 171 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met Human ABCC4 T796M peptide forms an unstable helix. Login to comment 172 ABCC4 p.Thr796Met ABCC4 p.Thr796Met A, percentage of ॷ-helix is plotted versus temperature (K) from the data derived from in silico molecular dynamics analysis of WT human ABCC4 and T796M mutant peptide. B and C, immunoblot of surface-biotinylated streptavidin-bound lysates (100 òe;g of protein/lane) prepared from NIH3T3 cells exposed to 37 or 28 &#b0;C for 24 h after 24 h of transfection with WT (human ABCC4) and T796M and its quantification. Login to comment 176 ABCC4 p.Thr796Met ABCC4 p.Thr796Met Next, we directly determined the effect of T796M mutation on the helix forming capabilities of a fragment of CL3(783-808), using circular dichroism spectrophotometry on CL3 peptides from human WT ABCC4 or T796M CL3 using approaches we previously reported (42). Login to comment 177 ABCC4 p.Thr796Met ABCC4 p.Thr796Met Next, we directly determined the effect of T796M mutation on the helix forming capabilities of a fragment of CL3(783-808), using circular dichroism spectrophotometry on CL3 peptides from human WT ABCC4 or T796M CL3 using approaches we previously reported (42). Login to comment 179 ABCC4 p.Thr796Met In contrast, the ABCC4 peptide harboring the ABCC4 T796M mutation required substantially higher concentrations of TFE to elicit comparable changes in helicity. Login to comment 180 ABCC4 p.Thr796Met ABCC4 p.Thr796Met In contrast, the ABCC4 peptide harboring the ABCC4 T796M mutation required substantially higher concentrations of TFE to elicit comparable changes in helicity. Login to comment 181 ABCC4 p.Thr796Met These results are compatible with our molecular dynamics simulations (Fig. 3A) and provide additional confirmation that the T796M mutation alters the helicity of this region of CL3 in ABCC4. Login to comment 182 ABCC4 p.Thr796Met We observed that the helical content of T804M was consistently lower than the ZF Abcc4 at temperatures greater than 301 K (Fig. 4A), results that are similar to T796M (Fig. 3A). Login to comment 183 ABCC4 p.Thr796Met We observed that the helical content of T804M was consistently lower than the ZF Abcc4 at temperatures greater than 301 K (Fig. 4A), results that are similar to T796M (Fig. 3A). Login to comment 185 ABCC4 p.Thr796Met The Abcc4 and Abcc4 T804M proteins each increased expression at 28 &#b0;C; however, for Abcc4 T804M the mature band c is difficult to resolve (Fig. 4B), and instead we observed a strong increase in protein, a result that is qualitatively similar to human T796M. Login to comment 186 ABCC4 p.Thr796Met The Abcc4 and Abcc4 T804M proteins each increased expression at 28 &#b0;C; however, for Abcc4 T804M the mature band c is difficult to resolve (Fig. 4B), and instead we observed a strong increase in protein, a result that is qualitatively similar to human T796M. Login to comment 198 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr Notably, disease-related point mutations in CFTR CL3 at positions S945L and H949Y (Fig. 5A) (44) affected maturation of CFTR (26). Login to comment 199 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC4 p.His798Tyr ABCC4 p.Ser794Leu Notably, disease-related point mutations in CFTR CL3 at positions S945L and H949Y (Fig. 5A) (44) affected maturation of CFTR (26). Login to comment 200 ABCC4 p.His798Tyr ABCC4 p.Ser794Leu Because these two amino acids are adjacent to Thr-796 in ABCC4 and mostly conserved with CFTR, we hypothesized that this region of CL3 might be generally important to ABCC protein stability. Using site-directed mutagenesis, we generated the analogous mutations in human ABCC4 (S794L and H798Y) and ZF Abcc4 (A802L and H806Y). Login to comment 201 ABCC4 p.Ser794Leu ABCC4 S794L and H796Y as well as ZF Abcc4 A802L and H806Y substitutions affected both protein expression and highly reduced the levels of the mature band c (Fig. 5, C and E). Login to comment 202 ABCC4 p.Thr796Met ABCC4 p.Ser794Leu ABCC4 S794L and H796Y as well as ZF Abcc4 A802L and H806Y substitutions affected both protein expression and highly reduced the levels of the mature band c (Fig. 5, C and E). Login to comment 203 ABCC4 p.Thr796Met Notably, the expression pattern of these mutants with reduced mature glycosylated band c is similar to ABCC4 T796M (Fig. 5E). Login to comment 206 ABCC4 p.Arg815Ala Mutation R815A in Human ABCC4 Cytoplasmic Region 3 (CL3) Destabilizes the Protein-Because CLs interact with NBDs in CFTR (26), we hypothesized that a mutation in an amino acid of CL3 in the "coupling helix" (a CL region near the NBD) might disrupt NBD and CL interdomain interactions producing an alteration in stability. Login to comment 207 ABCC4 p.Arg815Ala Mutation R815A in Human ABCC4 Cytoplasmic Region 3 (CL3) Destabilizes the Protein-Because CLs interact with NBDs in CFTR (26), we hypothesized that a mutation in an amino acid of CL3 in the "coupling helix" (a CL region near the NBD) might disrupt NBD and CL interdomain interactions producing an alteration in stability. Login to comment 208 ABCC4 p.Arg815Ala To investigate this we mutated Arg-815 to Ala. Login to comment 209 ABCC4 p.Arg815Ala ABCC4 p.Arg815Ala To investigate this we mutated Arg-815 to Ala. Login to comment 210 ABCC4 p.Thr796Met ABCC4 p.Arg815Ala ABCC4 p.Arg815Ala ABCC4 p.Arg815Ala The R815A mutation resulted in reduced ABCC4 expression and loss of mature glycosylated band c (Fig. 6, B and C). Login to comment 211 ABCC4 p.Thr796Met ABCC4 p.Arg815Ala ABCC4 p.Arg815Ala ABCC4 p.Arg815Ala The reduction in mature ABCC4 is consistent with the prediction that CL3 Arg-815 interacts with the NBD to ensure proper folding of the protein To test if reduced temperature enhanced expression of R815A, NIH3T3 cells were transiently transfected with ABCC4, T796M-ABCC4, or R815A-ABCC4 and then incubated at 28 &#b0;C. Login to comment 212 ABCC4 p.Arg815Ala The amount of R815A protein increased, which paralleled an increase in mature glycosylated protein band c (Fig. 6, B and C). Login to comment 214 ABCC4 p.Thr796Met However, in ZF and human ABCC4 (T796M, is the analogous position), protein FIGURE 4. Login to comment 215 ABCC4 p.Thr796Met However, in ZF and human ABCC4 (T796M, is the analogous position), protein FIGURE 4. Login to comment 237 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr Furthermore, we extended these studies to show that other mutations in CL3 (analogous to those in CFTR (S945L and H949Y)) that also disrupt its helical properties reduce protein expression. Login to comment 238 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr Furthermore, we extended these studies to show that other mutations in CL3 (analogous to those in CFTR (S945L and H949Y)) that also disrupt its helical properties reduce protein expression. Login to comment 239 ABCC4 p.Thr796Met ABCC4 p.Thr796Met The reduced amounts of mutant ABCC4 protein (T804M in zebrafish and T796M in humans) are not related to an impaired translation of the ABCC4 mRNA, which is almost identical among WT and T796M mRNA despite an apparent alteration in the energetics of mRNA folding. Login to comment 240 ABCC4 p.Thr796Met ABCC4 p.Thr796Met ABCC4 p.Thr796Met The reduced amounts of mutant ABCC4 protein (T804M in zebrafish and T796M in humans) are not related to an impaired translation of the ABCC4 mRNA, which is almost identical among WT and T796M mRNA despite an apparent alteration in the energetics of mRNA folding. Login to comment 241 ABCC4 p.Thr796Met It is likely that disrupted processing in the ER of ABCC4 is disrupted by the T796M substitution. Login to comment 243 ABCC4 p.Thr796Met Like the T796M for human and the orthologous T804M in zebrafish, these addi- FIGURE 5. Login to comment 244 ABCC4 p.Thr796Met Like the T796M for human and the orthologous T804M in zebrafish, these addi- FIGURE 5. Login to comment 248 ABCC4 p.Thr796Met ABCC4 p.His798Tyr ABCC4 p.Ser794Leu E, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 and ABCC4 mutants (T796M, S794L, and H798Y), and these blots were probed with anti-GFP antibody. Login to comment 249 ABCC4 p.Thr796Met ABCC4 p.His798Tyr ABCC4 p.Ser794Leu E, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 and ABCC4 mutants (T796M, S794L, and H798Y), and these blots were probed with anti-GFP antibody. Login to comment 256 ABCC4 p.Arg815Ala The increased formation of the mature c-form of ABCC4 R815A at the subphysiological temperature of 28 &#b0;C is consistent with the proposal that CL3/NBD1 interactions are necessary for proper folding of ABCC4. Login to comment 257 ABCC4 p.Thr796Met ABCC4 p.Arg815Ala The increased formation of the mature c-form of ABCC4 R815A at the subphysiological temperature of 28 &#b0;C is consistent with the proposal that CL3/NBD1 interactions are necessary for proper folding of ABCC4. Login to comment 258 ABCC4 p.Thr796Met In further support of ABCC4 CL3 mutants being misfolded, preliminary studies show that that two small molecules that reduce the activity of the ER unfolded protein response (phenylbutyrate (29) and glycerol (36)) selectively rescue expression of the ABCC4 CL3 T796M (data not shown). Login to comment 259 ABCC4 p.Thr796Met In cells harboring ABCC4, a T796M substitution, reduced amounts of the mature glycosylated form are observed. Login to comment 260 ABCC4 p.Thr796Met ABCC4 p.Thr796Met In cells harboring ABCC4, a T796M substitution, reduced amounts of the mature glycosylated form are observed. Login to comment 261 ABCC4 p.Thr796Met ABCC4 p.Thr796Met These findings, and the internal mislocalization of T796M, suggest that either ER glycosylation of ABCC4 is impaired or the protein is mostly degraded before maturation is complete. Login to comment 262 ABCC4 p.Thr796Met ABCC4 p.Thr796Met We show that T796M has reduced mature ABCC4 (c-form) coupled with decreased abundance at the plasma membrane. Login to comment 263 ABCC4 p.Thr796Met ABCC4 p.Thr796Met We favor the idea that the T796M substitution does not generally produce defective glycosylation of ABCC4 because plasma membrane localization and ABCC4 glycosyl maturation are mostly restored at reduced temperatures. Login to comment 264 ABCC4 p.Thr796Met However, we cannot rule out the possibility that the increased expression of T796M at subphysiological temperatures might be due, in part, to reduced protease activity (it seems unlikely that reduced protease activity modifies glycosylation). Login to comment 266 ABCC4 p.Arg815Ala A, representation of interdomain interaction between R815A of CL3 region and Asp-522 of NBD1 domain. Login to comment 267 ABCC4 p.Arg815Ala ABCC4 p.Arg815Ala A, representation of interdomain interaction between R815A of CL3 region and Asp-522 of NBD1 domain. Login to comment 268 ABCC4 p.Arg815Ala B, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 WT and ABCC4 mutant (R815A) and incubated at 37 or 28 &#b0;C for 24 h after 24 h of transfection. Login to comment 269 ABCC4 p.Thr796Met These findings buttress our molecular dynamics simulations showing that the region containing T796M has less thermal stability, a finding consistent with this region and with CL3`s reduced ability to form an ॷ-helix. Login to comment 270 ABCC4 p.Thr796Met These findings buttress our molecular dynamics simulations showing that the region containing T796M has less thermal stability, a finding consistent with this region and with CL3`s reduced ability to form an ॷ-helix. Login to comment 277 ABCC4 p.Arg815Ala Likewise, we show that the predicted contact between CL3 Arg-815 and NBD1 Asp-522 when disrupted by an R815A substitution reduces the amount of mature ABCC4. Login to comment 278 ABCC4 p.Arg815Ala Likewise, we show that the predicted contact between CL3 Arg-815 and NBD1 Asp-522 when disrupted by an R815A substitution reduces the amount of mature ABCC4. Login to comment