Transmembrane region of wild-type and mutant M13 coat proteins. Conformational role of beta-branched residues.

Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition. We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13. This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI). Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region. Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction. Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein. 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site. In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat. These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants.     

Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. A., Tamamizu, S., Butler, D. H., Vibat, C. R. T., Hung, B., and McNamee, M. G. (1996) Biochemistry 35, 14139-14148; Ortiz-Miranda, S. I., Lasalde, J. A., Pappone, P. A., and McNamee, M. G. (1997) J. Membr. Biol. 158, 17-30; Tamamizu, S., Lee, Y. H., Hung, B., McNamee, M. G., and Lasalde-Dominicci, J. A. (1999) J. Membr. Biol. 170, 157-164]. In this study, we introduce tryptophan substitutions at 12 positions (C412W, M415W, L416W, I417W, C418W, I419W, I420W, G421W, T422W, V423W, S424W, and V425W) along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated alpha-bungarotoxin binding ([(125)I]-alpha-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [(125)I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The alphaV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential alpha-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated alpha-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.     

Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups

The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [(3)H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.     

Tryptophan scanning mutagenesis in the TM3 domain of the Torpedo californica acetylcholine receptor beta subunit reveals an alpha-helical structure

We used tryptophan substitutions to characterize the beta M3 transmembrane domain (betaTM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the betaTM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [125I]-alpha-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the betaTM3 domain was consistent with an alpha-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed alpha-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica beta subunit.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.     

Design and structural analysis of an engineered thermostable chicken lysozyme.

A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with delta Tm = 3.3 degrees C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.     

Mutational Analysis of Bacterial NAD^＋-dependent DNA Ligase： Role of Motif IV in Ligation Catalysis

The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif IV in ligation catalysis, site-directed mutants were constructed in a bacterial NAD^＋-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues （D286, G287, V289 and K291） in motif IV had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.     

Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted alpha-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 +/- 4% alpha-helical structure at 22 degrees C that melts reversibly with T(m,WT) = 50 +/- 2 degrees C and van't Hoff enthalpy deltaH(v,WT)(Tm) = 18 +/- 2 kcal/mol. G15A substitution leads to an increased alpha-helical content of 42 +/- 4% and an increased T(m,G15A) = 57 +/- 2 degrees C, which corresponds to stabilization by delta deltaG(app) = +0.4 +/- 1.5 kcal/mol. G15P mutant has approximately 20% alpha-helical content at 22 degrees C and unfolds with low cooperativity upon heating to 90 degrees C. R23P and T45P mutants are fully unfolded at 0-90 degrees C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative alpha-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal alpha-helical content reaching 74%. Comparison with the maximal alpha-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.     

Structural stabilities of recombinant scombridae fish myoglobins

An expression system of recombinant myoglobins (Mb) of 3 scombridae fish species was constructed. The stability of these Mbs was compared with native Mbs purified from slow skeletal muscle. The addition of hemin during the cultivation of an Escherichia coli strain harboring a pGEX-2T expression vector was found to be necessary to prevent recombinant Mb from degrading and to attain its proper folding. The stabilities of recombinant Mbs were generally lower than those of native Mbs, partly due to the absence of post-translational modification. The alpha-Helical content of bullet tuna recombinant Mb at 10 degrees C was the lowest (29.0%) among the recombinant Mbs examined (the values for bluefin tuna and bigeye tuna Mbs being 34.8 and 35.5%, respectively). On the other hand, the stabilities of recombinant Mbs of bluefin tuna and bigeye tuna against denaturants (urea and guanidine hydrochloride) were found to be similar, whereas bullet tuna recombinant Mb exhibited the lowest stability among these Mbs. The pattern of temperature-dependent decrease in the alpha-helical content supported these results.     

Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+). This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS). Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site revertants were isolated from K18C and Y31C. The revertants were found to have mutations in helices I, IV, and VII.     

Use of monoclonal antibodies to identify four neutralization immunogens on a common cold picornavirus, human rhinovirus 14.

A collection of 35 mouse monoclonal antibodies, raised against human rhinovirus 14 (HRV-14), was used to isolate 62 neutralization-resistant mutants. When cross-tested against the antibodies in a neutralization assay, the mutants fell into four antigenic groups, here called neutralization immunogens: NIm-IA, -IB, -II, and -III. Sequencing the mutant RNA in segments corresponding to serotype-variable regions revealed that the amino acid substitutions segregated into clusters, which correlated exactly with the immunogenic groups (NIm-IA mutants at VP1 amino acid residue 91 or 95; NIm-II mutants at VP2 residue 158, 159, 161, or 162; NIm-III mutants at VP3 residue 72, 75, or 78; and NIm-IB mutants at two sites, either VP1 residue 83 or 85, or residue 138 or 139). Examination of the three-dimensional structure of the virus (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London], 317:145-153, 1985) revealed that each of the substitution clusters formed a protrusion from the virus surface, and the side chains of the substituted amino acids pointed outward. Moreover, four of the amino acid substitutions, which initially appeared to be anomalous because they were encoded well outside the cluster groups, could be traced to surface positions immediately adjacent to the appropriate viral protrusions. We conclude that three of the four antigens, NIm-IB, -II, and -III, are discontinuous. Thus, the amino acid substitutions in all 62 mutants fell within the proposed immunogenic sites; there was no evidence for alteration of any antigenic site by a distal mutation.     

Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase. Kinetic properties and oligomerization pattern of nine substitution mutants of V106.

Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer-tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern.     

Functional correlation between a novel amino acid insertion at codon 19 in the protease of human immunodeficiency virus type 1 and polymorphism in the p1/p6 Gag cleavage site in drug resistance and replication fitness

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.     

Combining mutations in HIV-1 protease to understand mechanisms of resistance

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.     

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution

To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB.     

Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis

Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to improve our understanding of the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria: dissociation of tetramers into folded monomers and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by globally fitting them to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild-type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to those of WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR, it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stabilities. The observed differences in stability among the disease-associated TTR variants highlight the complexity and heterogeneity of TTR amyloid disease, an observation that has important implications for the treatment of these maladies.     

Altered conductance and permeability of Cx40 mutations associated with atrial fibrillation

Gap junctions ensure the rapid propagation of the action potential throughout the myocardium. Three mutant forms of connexin40 (Cx40; A96S, M163V, and G38D), the primary component of the atrial gap junction channel, are associated with atrial fibrillation and retain the ability to form functional channels. We determined the biophysical properties of these mutant gap junctions in transiently transfected HeLa and N2A cells. All three mutants showed macroscopic junctional conductances over the range of 0.5 to 40 nS, and voltage dependences comparable to those of wild-type (WT) Cx40. However, the unitary conductance of G38D channels was ∼1.6-fold higher than that of WT Cx40 channels (∼220 vs. ∼135 pS), whereas the unitary conductances of the A96S and M163V mutants were similar to that of WT Cx40. Furthermore, the M163V and G38D channels exhibited approximately two- and approximately fivefold higher permeability to the anionic dye Lucifer yellow (LY) relative to K⁺ (LY/K⁺) compared with that of WT Cx40, whereas A96S LY transfer was similar to that of WT (G38D > M163V > A96S ≈ Cx40WT). In contrast, G38D channels were almost impermeable to cationic ethidium bromide (EtBr), suggesting that G38D alters channel selectivity. Conversely, A96S and M163V channels showed enhanced EtBr permeability relative to WT Cx40, with the following permeability order: M163V > A96S > Cx40WT > G38D. Altered conductive and permeability properties of mutant channels suggest an essential role for Cx40-mediated biochemical and electrical coupling in cardiac tissues. The altered properties of the three single-base substitution mutants may play a role in mechanisms of reentry arrhythmias.