Transmembrane-spanning Domains Research Articles

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Abstract 1621: PRAF2 knockdown inhibits cell proliferation and migration of neuroblastoma cells

Abstract Prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is a novel 19-kDa protein with four transmembrane-spanning domains that belongs the PRAF protein family that plays a role in vesicle trafficking and cargo transport. PRAF2 is strongly expressed in several cultured neuroblastoma (NB) cells (for example, SK-N-SH, SH-SY5Y, LAN-1). NB is a tumor that derived from the precursor cells of sympathetic nervous system and the most common extracranial solid tumor of childhood. Recently, our lab identified PRAF2 as a candidate prognostic marker of NB. In the present study, we examined the function of PRAF2 in cell cycle progression of human NB cells. In order to analyze the role of PRAF2, we generated a stable RNA interference (RNAi) short hairpin (sh) PRAF2 knockdown NB cell line (SK-N-SH shPRAF2). We observed that PRAF2 downregulation suppressed the cell viability of SK-N-SH cells. Moreover, PRAF2 knockdown also inhibited migration of SK-N-SH cells. Notably, PRAF2 downregulation also elevated the protein levels of cyclin-dependent kinase (Cdk) inhibitor p27Kip1 in these cells, therefore suggesting a role for PRAF2 in the regulation of NB cell cycle regulation and cell proliferation. In summary, our results suggest that reduction of PRAF2 protein level inhibits cell proliferation and migration in SK-N-SH cells that renders PRAF2 a promising target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1621.

Glutamate transporters

AD= : Alzheimer disease; ALS= : amyotrophic lateral sclerosis; AQP4= : aquaporin 4; EAAT= : excitatory amino acid transporter; GABA= : γ-aminobutyric acid; PD= : Parkinson disease; SLC= : solute carrier family; SOD1= : superoxide dismutase 1; TNF= : tumor necrosis factor; VGLUT= : vesicular glutamate transporter. Glutamate is the major excitatory neurotransmitter in the mammalian CNS and is critically involved in mechanisms of synaptic plasticity, memory, and neuronal or glial cell death. Glutamate is stored in synaptic vesicles by the action of vesicular glutamate transporters (VGLUTs) and, following its release, exerts its effects via inotropic and metabotropic receptors. The synaptic effects of glutamate are rapidly terminated by action of glutamate transporters (excitatory amino acid transporters [EAATs]) located on the plasma membrane of astrocytes and neurons. These 2 transporter families differ in many of their functional properties. Studies in knockout mice and other experimental models have provided insights into the physiologic role of VGLUTs and EAATs in normal glutamatergic transmission and their potential role in neurologic disorders. Alterations in the function or expression of EAATs have been implicated in the pathophysiology of stroke, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), Parkinson disease (PD), Huntington disease, HIV-associated dementia, malignant glioma, and other neurologic disorders. Loss of VGLUT has been reported in PD and AD. The physiology, regulation, and involvement of glutamate transporters in neurologic disease have been the subjects of several excellent reviews.1–10 ### Vesicular glutamate transporters. Glutamate is synthesized in the presynaptic terminals from glutamine by action of phosphate-activated glutaminase and is translocated into the lumen of presynaptic vesicles via VGLUTs3,5,6 (figure). VGLUT isoforms belong to the solute carrier family (SLC) SLC17 type I phosphate transporter family and are highly homologous, consisting of 12 putative transmembrane spanning domains. Glutamate uptake is driven by a proton (H+)-dependent electrochemical gradient across the vesicle membrane created by a vacuolar-type ATPase. Unlike other vesicular neurotransmitter transporters, the driving force for VGLUTs depends predominately on the existence of a vesicular membrane potential gradient, rather than a pH gradient. Figure Distribution and mechanisms of vesicular glutamate transporters (VGLUT) and excitatory amino acid transporters …

Investigation of Protonation Effects on ATP Binding in ABC Transporters

ATP binding cassette (ABC) transporters consist of two characteristic nucleotide binding domains (NBD) and two transmembrane-spanning domains (TMD). Binding and hydrolysis of ATP at the NBDs controls substrate transport or, in rare cases, other physiological functions.Previous studies have identified residue motifs of functional importance, but our understanding of the hydrolysis process in ABC NBDs remains incomplete. An acidic residue of the Walker-B motif has been suggested to act as a general base that abstracts a proton from the hydrolytic water. Other work has suggested a greater role of a histidine (in the switch motif) in activating the hydrolytic water. Also the role of the Mg2+ ion in orienting hydrolytic residues and waters is poorly understood.One limitation in interpreting existing ABC NBD structures for their hydrolytic function is the assignment of protonation states to the relevant residues and the exact orientation of water molecules in the NBDs. The highly charged nature of the NBS renders protonation assignment particularly challenging.In this study, we vary protonation states at the NBDs of the multidrug ABC exporter Sav1866 and simulate the ATP-bound NBD dimers by molecular dynamics. We consider combinations of protonation of the Walker-B glutamate, the switch histidine and the ATP itself with and without Mg2+. The resulting 24 systems are simulated to at least 50 ns duration.We show that the Mg2+ and residue protonation affect protein dynamics. Crucially. we show that the local geometries of ATP-binding residues in many available ABC NBD crystal structures can potentially be rationalized by different protonation states. Conformational changes of the Glu and His upon protonation support the idea of the Glu acting as a general base. Furthermore, we discuss the coordination and dynamics of putative hydrolytic waters.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1Acetylcholine Receptors Affects Plasma Membrane Expression

We investigated the functional role of a conserved motif, F(x)(6)LL, in the membrane proximal C-tail of the human muscarinic M(1) (hM(1)) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence (423)FRDTFRLLL(431). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [(3)H]N-methylscopolamine binding assays. The plasma membrane expression of hM(1) receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM(1) receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L(430) and L(431) play a role in folding hM(1) receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V(46) and L(47), that, when mutated, reduce the plasma membrane expression of hM(1) receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM(1) receptors to achieve a transport-competent state.

Expression, Solubilization and Purification of a Human Olfactory Receptor from Escherichia coli

Olfactory receptors pertaining to G protein-coupled receptor (GPCR) are integral membrane proteins composed of seven transmembrane spanning domains. It has been reported that these receptor proteins are difficult to overexpress, solubilize, and purify because of their complicated structures and strong hydrophobicity. In this study, full-length human olfactory receptor (hOR) 2AG1 was overexpressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene. This protein was difficult to solubilize with detergents and chaotropic agents, and only N-lauroyl sarcosine was found to be suitable for solubilizing it. In contrast, Triton X-100 was found to solubilize most of the impurity proteins from the insoluble fraction in E. coli. Based on this observation, we applied a simple and efficient column-free method using these two detergents for the purification of the olfactory receptor protein. In this method, the insoluble fraction of the cell lysate was first treated with Triton X-100 to remove impurity proteins. The remaining insoluble fraction was then further treated with N-lauroyl sarcosine to solubilize the olfactory receptor protein. Milligram quantity of the human olfactory receptor was produced. This is the first report to produce full-length of the olfactory receptor from E. coli.

Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.

Molecular cloning, gene expression, and tissue distribution of adiponectin and its receptors in the Japanese monkey, Macaca fuscata

Adiponectin is an adipocyte-derived hormone that affects regulation of metabolic syndrome such as insulin resistance, type-2 diabetes, and obesity. It functions via seven transmembrane domain receptors [i.e.,adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2)] that have been scarcely investigated in non-human primates. Molecular cloning of cDNAs for adiponectin, AdipoR1, and AdipoR2 that included the whole protein-coding region in the Japanese monkey, Macaca fuscata, was carried out. Tissue-specific expression of respective genes was analyzed with Northern blot hybridization. The essential Cys36 and four lysine residues in adiponectin, and transmembrane-spanning domains in AdipoR1 and AdipoR2 appear well conserved. While adiponectin mRNA is expressed only in adipose tissues, AdipoR1 mRNA was found to be expressed in various tissues including the brain. These results significantly add to the understanding of the molecular basis of obesity-related adipokines and their receptors in nonhuman primates.

Using Correlation Analysis To Predict Pairs Of Energetically Coupled Residues At The NBD-TMD Interface In CFTR

CFTR, whose failure causes cystic fibrosis, is a chloride channel, which belongs to the ATP binding cassette (ABC) transporter family. Like other ABC proteins, CFTR consists of two halves, each containing a cytosolic nucleotide-binding domain (NBD1, NBD2) and a transmembrane spanning domain (TMD1, TMD2). ATP binding and hydrolysis at the NBDs control the CFTR gate, presumed to be in the TMDs. It remains unclear precisely how the NBD/TMD coupling is mediated.We used correlation analysis to identify possible pairs of energetically coupled residues which might mediate direct interactions between the NBDs and the TMDs. First, since CFTR belongs to a subgroup of ABCs, which contain two very divergent NBDs, we constructed an alignment containing only similar, asymmetric transporters. We implemented 5 different correlation algorithms. The major difficulty with correlation analysis is the prevalence of false positives due to non-independence of sequences resulting from evolutionary constraints. Only one algorithm corrects for non-independence by referring to an inferred phylogenetic tree, resulting in a smaller output. The other 4 methods assume independence and assign a score to every possible pair of alignment positions. Only pairs scoring in the top 1% were included.To select candidate pairs at the NBD/TMD interface we looked for pairs found with more than one algorithm and separated by less than ∼15A on a Sav1866 based homology model of CFTR. We also checked the distributions of amino acids at the two positions on the inferred phylogenetic tree, to eliminate correlations clearly due to evolutionary branching. We identified several pairs which are close to sites which can be cross-linked after cysteine substitution. Functional characterization of the effects of mutations on single-channel kinetics is underway.

Tas1R1–Tas1R3 taste receptor variants in human fungiform papillae

Monosodium glutamate as well as metabotropic and ionotropic glutamate receptor agonists have been reported to be perceived as umami by humans. In spite of the fact that Tas1R1–Tas1R3 has been shown to mediate most of the glutamate taste sensation in mice other candidate receptors have been put forward for which a clear role in detection is still lacking. This work was aimed at investigating the molecular determinants underlying umami taste detection in humans. First, we show evidence supporting expression of Tas1R1 and Tas1R3 but not mGluRs in the fungiform papillae of several individuals. Next, we report a number of naturally occurring l-glutamate taste receptor variants and their frequency in a population of Caucasian subjects. Detailed analysis of 9 non-synonymous single nucleotide polymorphisms from three l-glutamate taste GPCR candidates uncovers receptor specific clusters such that all substitutions in Tas1R1 are located in the extracellular N-terminal ligand-binding domain while in Tas1R3 they mostly affect residues in the seven transmembrane-spanning core domain responsible for the interaction with antagonists and allosteric modulators. In mGluR1, nsSNPs identified are clustered in the intracellular C-terminal tail, which is thought to play a role in signaling. Taken together, these results suggest that Tas1R1–Tas1R3 receptor variants found in human fungiform papillae might contribute to inter-individual differences of sensitivity to l-glutamate.

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

Identification of an Evolutionarily Conserved Extracellular Threonine Residue Critical for Surface Expression and Its Potential Coupling of Adjacent Voltage-sensing and Gating Domains in Voltage-gated Potassium Channels

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

The SLC16 monocaboxylate transporter family

1. The monocarboxylate transporter (MCT, SLC16) family comprises 14 members, of which to date only MCT1–4 have been shown to carry monocarboxylates, transporting important metabolic compounds such as lactate, pyruvate and ketone bodies in a proton-coupled manner. The transport of such compounds is fundamental for metabolism, and the tissue locations, properties and regulation of these isoforms is discussed.2. Of the other members of the MCT family, MCT8 (a thyroid hormone transporter) and TAT1 (an aromatic amino acid transporter) have been characterized more recently, and their physiological roles are reviewed herein. The endogenous substrates and functions of the remaining members of the MCT family await elucidation.3. The MCT proteins have the typical twelve transmembrane-spanning domain (TMD) topology of membrane transporter proteins, and their structure–function relationship is discussed, especially in relation to the future impact of the single nucleotide polymorphism (SNP) databases and, given their ability to transport pharmacologically relevant compounds, the potential impact for pharmacogenomics.

Alternative splicing and exon duplication generates 10 unique porcine 5-HT4 receptor splice variants including a functional homofusion variant

5-HT(4) receptors are present in human and porcine atrial myocytes while they are absent from the hearts of small laboratory animals. The pig is therefore the only available nonprimate animal model in which to study cardiac 5-HT(4) receptor function under physiological conditions. While several human splice variants of the 5-HT(4) receptor have been described, the splicing behavior of this receptor in porcine tissue is currently unknown. Here we report on the identification of nine novel COOH-terminal splice variants of the porcine 5-HT(4) receptor, which were named 5-HT(4(b2, j, k, l, m, o, p, q, r)). The internal h-variant was found in combination with several COOH-terminal exons. In addition, splice variants were found that comprised duplicated exons fused to the common region of the 5-HT(4) receptor, thereby providing evidence for a duplication of the porcine HTR4 gene. One of these variants putatively encoded a nine transmembrane-spanning domain homofusion receptor, 5-HT(4(9TM)); also the other variants with a duplicated region might translate into functional, transcriptionally fused dimeric 5-HT(4) receptor variants. The elucidation of the genomic context confirmed that the variants were not genomic artefacts but originated from alternative splicing. This was further corroborated by a functional analysis of the variants 5-HT(4(a)), 5-HT(4(r)), and 5-HT(4(9TM)). To our knowledge, our data are the first to report on a functional GPCR with more than seven predicted transmembrane domains. These findings urge for caution when interpreting data on 5-HT(4) receptor-related pharmacology obtained in the pig; validation at the molecular level might be needed before extrapolating results to human.

Sodium‐induced allosteric changes in D2 dopamine receptor local dynamics modulate ligand selectivity

The basis of allostery in GPCRs and its implications for ligand recognition remain a mystery. While determining the source of large differences in measured ligand affinity reported for a mutant D2 dopamine receptor, we uncovered a unique and significant allosteric response to the physiological modulator sodium. This response is characterized by a sodium‐dependent enhanced affinity for ligands of a specific structural class having either agonist or antagonist functional properties. To explore possible mechanisms for our empirically‐derived results, the influence of sodium on D2 receptor dynamics was examined by computational analysis of the molecular normal modes of motion. This result revealed divergence in the component motions of the D2 receptor with and without sodium bound to the putative sodium binding regions. One particularly sensitive mode involved the concerted motions of the extracellular portions of transmembrane‐spanning domains, which when computed in the presence of sodium appeared to disrupt a favorable pi‐stacking interaction between TM2 and TM3 freeing an aromatic residue in the receptor model for favorable interactions with aromatic portions of the ligands increasing their affinity. This demonstrates how a sodium‐dependent change in dynamic properties can yield the experimentally observed allosteric phenomenon.Supported R01 MH063162 (JAS), P01 DA012923 and K05 DA00060 (HW).

Progesterone Acts via Progesterone Receptors A and B to Regulate Breast Cancer Resistance Protein Expression: Fig. 1.

The breast cancer resistance protein (BCRP; ABCG2) is an ATP-dependent efflux multidrug transporter that belongs to the G family of half-transporters that consist of six transmembrane-spanning domains and must homodimerize to form the active membrane transporter. It is expressed in the apical plasma membrane domain of the small intestine, endothelium, and liver, where it has been shown to play an important role in limiting drug absorption and distribution and in enhancing drug clearance, respectively. BCRP is also expressed in the apical membrane of mammary alveolar epithelia, where it mediates efflux of substrates into milk, and in the placental syncytiotro-phoblasts, where it reduces fetal exposure to these substrates. BCRP substrates include numerous drugs (topotecan, nitrofurantoin, cimetidine) as well as food carcinogens (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and the vitamins riboflavin and folic acid. BCRP expression is regulated by a number of nuclear transcription factors, including the peroxisome proliferator-activated receptor-gamma and Hif-1. This issue of Molecular Pharmacology includes a study (p. 845) now conclusively demonstrating that progesterone acts via the progesterone A and B receptors to regulate BCRP expression in a placental cell line.

Charge scan reveals an extended region at the intracellular end of the GABA receptor pore that can influence ion selectivity.

Selective permeability is a fundamental property of ion channels. The Cys-loop receptor superfamily is composed of both excitatory (ACh, 5-HT) and inhibitory (GABA, glycine) neurotransmitter-operated ion channels. In the GABA receptor, it has been previously shown that the charge selectivity of the integral pore can be altered by a single mutation near the intracellular end of the second transmembrane-spanning domain (TM2). We have extended these findings and now show that charge selectivity of the anionic ρ1 GABA receptor can be influenced by the introduction of glutamates, one at a time, over an 8–amino acid stretch (−2′ to 5′) in the proposed intracellular end of TM2 and the TM1–TM2 intracellular linker. Depending on the position, glutamate substitutions in this region produced sodium to chloride permeability ratios (PNa+/Cl−) varying from 0.64 to 3.4 (wild type PNa+/Cl− = 0). In addition to providing insight into the mechanism of ion selectivity, this functional evidence supports a model proposed for the homologous nicotinic acetylcholine receptor in which regions of the protein, in addition to TM2, form the ion pathway.

A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse

Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.