Residues In Transmembrane Domains Research Articles

Mutational and pH Analysis of Ionic Residues in Transmembrane Domains of Vesicular Acetylcholine Transporter

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.

Ligand binding to the human MT2 melatonin receptor: The role of residues in transmembrane domains 3, 6, and 7

To better understand the mechanism of interactions between G-protein-coupled melatonin receptors and their ligands, our previously reported homology model of human MT2 receptor with docked 2-iodomelatonin was further refined and used to select residues within TM3, TM6, and TM7 potentially important for receptor–ligand interactions. Selected residues were mutated and radioligand-binding assay was used to test the binding affinities of hMT2 receptors transiently expressed in HEK293 cells. Our data demonstrate that residues N268 and A275 in TM6 as well as residues V291 and L295 in TM7 are essential for 2-iodomelatonin binding to the hMT2 receptor, while TM3 residues M120, G121, V124, and I125 may participate in binding of other receptor agonists and/or antagonists. Presented data also hint at possible specific interaction between the side-chain of Y188 in second extracellular loop and N-acetyl group of 2-iodomelatonin.

Opioid Receptor-Like 1 (ORL1) Molecular “Road Map” to Understanding Ligand Interaction and Selectivity

The opioid receptor-like 1 (ORL1) system has attracted a lot of attention owing to its diverse physiological role and by its close structural proximity toward the classical opioid receptors. Even though they share a close sequence similarity, the ligand recognition pattern for the ORL1 receptor and the classical opioid receptors remains highly distinct. In addition, functional diversification observed between the ORL1 receptor system and classical opioid receptors clearly indicates that subtle changes in the structural makeup of a receptor are enough to delineate them. A clear understanding of the structural requirements for ligand selectivity by classical opioid receptors and identification of a common "opioid binding pocket" has not been achieved yet. At this juncture, the ORL1 receptor system presents itself as a potential tool in the quest for elucidating critical elements directing ligand selectivity. The current paper is a compilation of several site-directed mutagenesis studies conducted on the ORL1 receptor system. The mutagenesis studies concentrated on the transmembrane domain residues are reported with the changes observed in terms of both binding and functional activation of the receptor. Given the critical role played by this G-protein coupled receptor, molecular level understanding of this ORL1 receptor system would aid in rational design and development of agonists and antagonists with multiple therapeutic applications.

Role of third extracellular domain of plasma membrane Ca 2+–Mg 2+-ATPase based on the novel inhibitor caloxin 3A1

The plasma membrane Ca 2+ pump (PMCA) is a Ca 2+–Mg 2+-ATPase that expels Ca 2+ from cells to help them maintain low concentrations of cytosolic Ca 2+ ([Ca 2+] i). It contains five putative extracellular domains (PEDs). Earlier we had reported that binding to PED2 leads to PMCA inhibition. Mutagenesis of residues in transmembrane domain 6 leads to loss of PMCA activity. PED3 connects transmembrane domains 5 and 6. PED3 is only five amino acid residues long. By screening a phage display library, we obtained a peptide sequence that binds this target. After examining a number of peptides related to this original sequence, we selected one that inhibits the PMCA pump (caloxin 3A1). Caloxin 3A1 inhibits PMCA but not the sarcoplasmic reticulum Ca 2+-pump. Caloxin 3A1 did not inhibit formation of the 140 kDa acylphosphate intermediate from ATP or its degradation. Thus, PEDs play a role in the reaction cycle of PMCA even though sites for binding to the substrates Ca 2+ and Mg-ATP 2−, and the activator calmodulin are all in the cytosolic domains of PMCA. In endothelial cells exposed to low concentration of a Ca 2+-ionophore, caloxin 3A1 caused a further increase in [Ca 2+] i proving its ability to inhibit PMCA pump extracellularly. Thus, even though PED3 is the shortest PED, it plays key role in the PMCA function.

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity.

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.

Dimerization of the Transmembrane Domain of Integrin αIIb Subunit in Cell Membranes

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

Nonrandom intragenic variations in patterns of codon bias implicate a sequential interplay between transitional genetic drift and functional amino acid selection.

Although most codon third bases appear to be functionless, the synonymous codons so defined exhibit a strikingly nonrandom distribution (codon bias) within human and other genes. To examine this phenomenon further, we generated a database of DNA sequences encoding human transmembrane cell-surface receptor proteins. Using this database we show here that the guanine and cytosine content of codon third bases (GC3) varies intragenically with the nature of the specified receptor domains (transmembrane > extracellular > intracellular domains; p < 0.001), the phenotype of the encoded amino acids (hydrophobic > hydrophilic > neutral amino acids; p < 0.001), and the receptor affiliation of the transmembrane (G-protein-coupled receptors > receptor tyrosine kinases; p < 0.001). Within gene regions specifying transmembrane domains, GC3 declines as domain functionality becomes redundant with increasing hydrophobicity (p < 0.001). Codons containing the second-base cytosine (XCZ, which encodes neutral amino acids) are selectively depleted of third-base adenine content (A3: XCA codons) when encoding transmembrane domain residues, consistent with positive selection for transitional mutation of XCG to XTG (which encodes hydrophobic amino acids) rather than to the synonymous XCA. Supporting this XCG --> XTG mechanism of codon bias, the G3:A3 ratio of codons specifying the transmembrane amino acid glycine (GGZ) is intermediate between that of its functional homolog alanine (GCZ) and that of hydrophobic valine (GTZ), even though the C3:T3 ratios are similar. Conversely, nearest-neighbor analysis of third bases 5' to codons specifying valine and leucine (CTZ) confirms a significant difference in C3:T3 but not G3:A3 ratios (i.e., C3/G1 --> T3/G1 > C3/A1; p < 0.001), consistent with the functionally advantageous retention of hydrophobic residues. These data raise the possibility that patterns of intragenic codon bias reflect a balance between negative and positive selection, suggesting in turn that analysis of codon third-base usage may help to predict the functional significance of encoded products.

Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes.

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Deltarer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.

Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6

Mutations at natural resistance-associated macrophage protein 1 (Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal transporter 1 [DMT1]) affect iron homeostasis and cause severe microcytic anemia. Structure-function relationships in the Nramp superfamily were studied by mutagenesis, followed by functional characterization in yeast and in mammalian cells. These studies identify 3 negatively charged and highly conserved residues in transmembrane domains (TM) 1, 4, and 7 as essential for cation transport by Nramp2/DMT1. The introduction of a charged residue (Gly185Arg) in TM4 found in the naturally occurring microcytic anemia mk (mouse) and Belgrade (rat) mutants is shown to cause a partial or complete loss of function in mammalian and yeast cells, respectively. A pair of mutation-sensitive and highly conserved histidines (His267, His272) was identified in TM6. Surprisingly, inactive His267 and His272 mutants could be rescued by lowering the pH of the transport assay. This indicates that His267/His272 are not directly involved in metal binding but, rather, play an important role in pH regulation of metal transport by Nramp proteins.

Palmitoylation of Carboxypeptidase D: IMPLICATIONS FOR INTRACELLULAR TRAFFICKING

Covalent lipid modifications mediate protein-membrane and protein-protein interactions and are often essential for function. The purposes of this study were to examine the Cys residues of the transmembrane domain of metallocarboxypeptidase D (CPD) that could be a target for palmitoylation and to clarify the function of this modification. CPD is an integral membrane protein that cycles between the trans Golgi network and the plasma membrane. We constructed AtT-20 cells stably expressing various constructs carrying a reporter protein (albumin) fused to a transmembrane domain and the CPD cytoplasmic tail. Some of the constructs contained the three Cys residues present in the CPD transmembrane region, while other constructs contained Ala in place of the Cys. Constructs carrying Cys residues were palmitoylated, while those constructs lacking the Cys residues were not. Because palmitoylation of several proteins affects their association with cholesterol and sphingolipid-rich membrane domains or caveolae, we tested endogenous CPD and several of the reporter constructs for resistance to extraction with Triton X-100. A construct containing the Cys residues of the CPD transmembrane domain was soluble in Triton X-100 as was endogenous palmitoylated CPD, indicating that palmitoylation does not target CPD to detergent-resistant membrane rafts. Interestingly, constructs of CPD that lack palmitoylation sites have an increased half-life, a slightly more diffuse steady-state localization, and a slower rate of exit from the Golgi as compared with constructs containing palmitoylation sites. Thus, the covalent attachment of palmitic acid to the Cys residues of CPD has a functional significance in the trafficking of the protein.

Thermostability of membrane protein helix–helix interaction elucidated by statistical analysis

A prerequisite for the survival of (micro)organisms at high temperatures is an adaptation of protein stability to extreme environmental conditions. In contrast to soluble proteins, where many factors have already been identified, the mechanisms by which the thermostability of membrane proteins is enhanced are almost unknown. The hydrophobic membrane environment constrains possible stabilizing factors for transmembrane domains, so that a difference might be expected between soluble and membrane proteins. Here we present sequence analysis of predicted transmembrane helices of the genomes from eight thermophilic and 12 mesophilic organisms. A comparison of the amino acid compositions indicates that more polar residues can be found in the transmembrane helices of thermophilic organisms. Particularly, the amino acids aspartic acid and glutamic acid replace the corresponding amides. Cysteine residues are found to be significantly decreased by about 70% in thermophilic membrane domains suggesting a non-specific function of most cysteine residues in transmembrane domains of mesophilic organisms. By a pair-motif analysis of the two sets of transmembrane helices, we found that the small residues glycine and serine contribute more to transmembrane helix–helix interactions in thermophilic organisms. This may result in a tighter packing of the helices allowing more hydrogen bond formation.

Substrate specificity of nickel/cobalt permeases: insights from mutants altered in transmembrane domains I and II.

HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. Two histidine residues in a Ni2+ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co2+ and Ni2+ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+ uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+ and Co2+ with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+ uptake capacity and conferred to the variant the ability to transport Co2+. NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.

The role of transmembrane helix 5 in agonist binding to the human H3 receptor.

We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [(125)I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-alpha-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.

Polar mutations in membrane proteins as a biophysical basis for disease.

Transmembrane (TM) alpha-helices are surrounded by the hydrocarbon chains of the lipid bilayer. The low dielectric constant of this environment makes it extremely unfavorable for a residue with a polar side chain to exist in a non-H-bonded state. Therefore, in combination with a wild-type polar residue partner, a polar TM mutant could generate, in some cases, a non-native H-bond that could impair native protein structure/function-and possibly lead to a disease state. We have examined protein mutation databases and have found many examples of TM-based apolar to polar mutations that are, in fact, a cause of human disease. Here we review the various molecular defects that such mutations can produce, including impeding protein dynamics by side-chain-side-chain interhelical H-bond cross-links; alteration of helical packing through steric hindrance; and disruption of a protein active site. We further note that the reverse case--membrane-embedded polar to apolar mutations--can similarly cause human disease, implying that native interhelical H-bonds can also play pivotal roles in stabilizing native TM domains. As a specific example, we show that the Gly to Arg mutation occurs statistically more frequently in TM domains as compared to its occurrence in soluble domains, suggesting that TM-based G-to-R mutations have a high "phenotypic propensity" for disease. A more complete understanding of how mutations involving polar residues in TM domains of proteins translate into compromised function may aid in the development of novel therapeutics.

Tyrosine residue 271 of the norepinephrine transporter is an important determinant of its pharmacology

The aim was to examine the functional importance in the norepinephrine transporter (NET) of (i) the phenylalanine residue at position 531 in transmembrane domain (TMD) 11 by mutating it to tyrosine in the rat (rF531Y) and human (hF531Y) NETs and (ii) the highly conserved tyrosine residues at positions 249 in TMD 4 of human NET (hNET) (mutated to alanine: hY249A) and 271 in TMD 5, by mutating to alanine (hY271A), phenylalanine (hY271F) and histidine (hY271H). The effects of the mutations on NET function were examined by expressing the mutant and wildtype NETs in COS-7 cells and measuring the K m and V max for uptake of the substrates, [ 3H]norepinephrine, [ 3H]MPP + and [ 3H]dopamine, the K D and B max for [ 3H]nisoxetine binding and the K i of the inhibitors, nisoxetine, desipramine and cocaine, for inhibition of [ 3H]norepinephrine uptake. The K m values of the substrates were lower for the mutants at amino acid 271 than hNET and unaffected for the other mutants, and each mutant had a significantly lower V max than NET for substrate uptake. The mutations at position 271 caused an increase in the K i or K D values of nisoxetine, desipramine and cocaine, but there were no effects for the other mutations. Hence, the 271 tyrosine residue in TMD 5 is an important determinant of NET function, with the mutants showing an increase in the apparent affinities of substrates and a decrease in the apparent affinities of inhibitors, but the 249 tyrosine and 531 phenylalanine residues do not have a major role in determining NET function.

The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents.

Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.

Conformationally Sensitive Residues in Transmembrane Domain 9 of the Na+/dicarboxylate Co-transporter

The Na(+)/dicarboxylate co-transporter, NaDC-1, couples the transport of sodium and Krebs cycle intermediates, such as succinate and citrate. Previous studies identified two functionally important amino acids, Glu-475 and Cys-476, located in transmembrane domain (TMD) 9 of NaDC-1. In the present study, each amino acid in TMD-9 was mutated to cysteine, one at a time, and the accessibility of the membrane-impermeant reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) to the replacement cysteines was determined. Cysteine substitution was tolerated at all but five of the sites: the A461C mutant was not present at the plasma membrane, whereas the F473C, T474C, E475C, and N479C mutants were inactive proteins located on the plasma membrane. Cysteine substitution of four residues found near the extracellular surface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in proteins that were sensitive to inhibition by MTSET. The accessibility of MTSET to the four substituted cysteines was highest in the presence of the transported cations, sodium or lithium, and low in choline. The four mutants also exhibited substrate protection of MTSET accessibility. The MTSET accessibility to S478C, A481C, and A480C was independent of voltage. In contrast, T482C was more accessible to MTSET in choline buffer at negative holding potentials, but there was no effect of voltage in sodium buffer. In conclusion, TMD-9 may be involved in transducing conformational changes between the cation-binding sites and the substrate-binding site in NaDC-1, and it may also form part of the translocation pathway through the transporter.

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp 90 residue

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp 90 of the catfish GnRH receptor can be substituted by a neutral Asn 90 without abolishing receptor signalling if another negatively charged Glu 93 is introduced in a proximal region of the receptor interior, thereby mimicking the Glu 90–Lys 121 salt bridge of mammalian GnRH receptors.

Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier.

The reduced folate carrier (RFC1) plays a major role in the delivery of folates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane domains. This article explores the role of these residues in transport function by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines transfected with R131L, R155L, or R366L all lacked activity, despite high levels of protein expression in the plasma membrane, suggesting the crucial role of these amino acid residues in RFC1 function. In several mutant carriers, R26L, R42L, and K332L, there was little or no change in the influx K(t) value for MTX or influx K(i) value for folic acid. However, the R26L, R42L, and K332L carriers had decreased affinity for reduced folates. This was most prominent for K404L, which had 11- and 4-fold increases in influx K(i) for 5-methyl-THF and 5-formyl-THF, respectively, compared with L1210 cells. The marked influx stimulation observed with wild-type carrier when extracellular chloride was decreased was significantly diminished when influx was mediated by the K404L carrier, but was only slightly decreased with the R26L, R42L, and K332L mutants. This suggested that the K404 residue may be a major site of inhibition by chloride in the wild-type carrier. These studies indicate the important role that some positively charged residues within transmembrane domains of RFC1 play in RFC1 function.

Update of the calcium-sensing receptor and calcimimetics

The extracellular calcium-sensing receptor (CaR) is a seven transmembrane–spanning G-protein–coupled receptor. It contains a large extracellular domain (ECD), the signature seven transmembrane domains (TMDs), three intracellular loops, and a large carboxy terminal tail. The CaR is expressed in the parathyroid, thyroid C-cells, kidney, brain, pituitary, intestine, bone marrow, skin, and other tissues. CaRs regulate serum calcium homeostasis through changes in parathyroid hormone (PTH) levels and renal calcium excretion. CaRs may also participate in controlling cell growth and differentiation, and in augmenting the actions of other hormones. Autosomal dominant hypocalcemia is caused by gain-of-function mutations that increase the sensitivity of the CaR to Ca2+. Familial benign hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism result from inactivating mutations of the CaR. The locations of the point mutations that are responsible for both increased and decreased sensitivity to extracellular Ca2+ are typically in the ECD of the CaR, supporting the idea that the ECD is responsible for both Ca2+ binding and sensing changes in Ca2+. Binding of Ca2+ to the ECD may cause a conformational change in the ECD that promotes an interaction with critical TMD residues, thus enhancing G-protein–dependent signal transduction. A class of compounds called calcimimetics potentiates the effects of extracellular Ca2+ on the CaR. Calcimimetics have been shown to reduce PTH and serum Ca2+ in patients with primary (1°) and secondary (2°) hyperparathyroidism (HPT). Studies with rats suggest that calcimimetics may be useful in treating renal osteodystrophy and may also suppress parathyroid growth. Further long-term studies will be needed to address the possible benefit of calcimimetics to patients with 1° and 2° HPT.