Fourth Transmembrane Domain Research Articles

An intramembrane glutamic acid governs peripherin/rds function for photoreceptor disk morphogenesis.

Peripherin/rds (P/rds), the product of the retinal degeneration slow (rds) gene, is a tetraspanin protein that plays a pivotal role for photoreceptor outer segment (OS) structure and is involved in a broad spectrum of inherited retinal degenerations. P/rds interacts with the homologous protein rom-1, previously proposed to regulate P/rds function. The authors examined the significance of an intramembrane glutamic acid conserved in all P/rds proteins (and many other tetraspanins) but absent in all rom-1 orthologs. The authors performed isosteric glutamine substitution of the conserved glutamate at position 276, in the fourth transmembrane domain of bovine P/rds, and expressed E276Q P/rds in COS-1 cells and in transgenic mouse photoreceptors of rds +/+, -/+, and -/- backgrounds. Western blot, immunoprecipitation, and sedimentation analyses were used to assess protein structure and interactions. Microscopy and electroretinography were used to characterize transgenic protein localization and retinal photoreceptor structure and function. E276Q P/rds was expressed, assembled, and properly localized in photoreceptor OSs of transgenic mice. In contrast to wild-type (WT) P/rds, however, this mutant did not rescue the OS structural defects observed in rds -/- and -/+ mice. Moreover, E276Q expression did not prevent the retinal degeneration that occurred as a consequence of OS disruption. E276 plays a critical role in P/rds support of photoreceptor OS structure. This finding provides a molecular rationale for asymmetry in P/rds and rom-1 function and for rom-1 regulation of P/rds activity. These findings also suggest that ionizable intramembrane residues may serve regulatory roles for tetraspanin proteins more generally.

Kinetic Relationship between the Voltage Sensor and the Activation Gate in spHCN Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels.

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3-17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

Slow Conformational Changes of the Voltage Sensor during the Mode Shift in Hyperpolarization-Activated Cyclic-Nucleotide-Gated Channels

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are activated by hyperpolarizations that cause inward movements of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. If HCN channels are held open for prolonged times (>50 ms), HCN channels undergo a mode shift, which in sea urchin (spHCN) channels induces a >50 mV shift in the midpoint of activation. The mechanism underlying the mode shift is unknown. The mode shift could be attributable to conformational changes in the pore domain that stabilize the open state of the channel, which would indirectly shift the voltage dependence of the channel, or attributable to conformational changes in the voltage-sensing domain that stabilize the inward position of S4, thereby directly shifting the voltage dependence of the channel. We used voltage-clamp fluorometry to detect S4 movements and to correlate S4 movements to the different activation steps in spHCN channels. We here show that fluorophores attached to S4 report on fluorescence changes during the mode shift, demonstrating that the mode shift is not simply attributable to a stabilization of the pore domain but that S4 undergoes conformational changes during the mode shift. We propose a model in which the mode shift is attributable to a slow, lateral movement in S4 that is triggered by the initial S4 gating-charge movement and channel opening. The mode shift gives rise to a short-term, activity-dependent memory in HCN channels, which has been shown previously to be important for the stable rhythmic firing of pacemaking neurons and could significantly affect synaptic integration.

Penumbra Encodes a New Tetraspanin Critical to Effective Erythropoiesis.

To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbra was identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine Penumbra mRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbra as a probe, we identified the human Penumbra from a human bone marrow cDNA library. The human Penumbra also encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbra gene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbra deletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neo selectable marker, the targeting vector employed a self-excising Cre-Neo cassette driven by a testis-specific promoter. At 3 months of age, Penumbra−/− mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra−/− mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra−/− mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p = 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p = 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/−- mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbra plays an important role in erythropoiesis and suggest that Penumbra may be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias.

CaMK‐II modulation of GABAAreceptors expressed in HEK293, NG108‐15 and rat cerebellar granule neurons

The gamma-aminobutyric acid type A (GABA(A)) receptor is a pentameric ligand-gated ion channel responsible for fast synaptic inhibition in the brain. Phosphorylation of the GABA(A) receptor by serine/threonine protein kinases, at residues located in the intracellular loop between the third and fourth transmembrane domains of each subunit, can dynamically modulate receptor trafficking and function. In this study, we have assessed the effect that Ca(2+)-calmodulin-dependent protein kinase-II (CaMK-II) has on GABA(A) receptors. The intracellular application of preactivated CaMK-II failed to modulate the function of alphabeta and alphabetagamma subunit GABA(A) receptors heterologously expressed in human embryonic kidney (HEK)293 cells. However, application of similarly preactivated alpha-CaMK-II significantly potentiated the amplitudes of whole-cell GABA currents recorded from rat cultured cerebellar granule neurons and from recombinant GABA(A) receptors expressed in neuroblastoma, NG108-15, cells. The modulation by alpha-CaMK-II of current amplitude depended upon the subunit composition of GABA(A) receptors. alpha-CaMK-II potentiated GABA currents recorded from alpha1beta3 and alpha1beta3gamma2 GABA(A) receptors, but was unable to functionally modulate beta2 subunit-containing receptors. Similar results were obtained from beta2 -/- mouse cerebellar granule cell cultures and from rat granule cell cultures overexpressing recombinant alpha1beta2 or alpha1beta3 GABA(A) receptors. alpha-CaMK-II had a greater effect on the modulation of GABA responses mediated by alpha1beta3gamma2 compared with alpha1beta3 receptors, indicating a possible role for the gamma2 subunit in CaMK-II-mediated phosphorylation. In conclusion, CaMK-II can upregulate the function of GABA(A) receptors expressed in neurons or a neuronal cell line that is dependent on the beta subunit co-assembled into the receptor complex.

Mass Spectrometric Analysis of Glycine Receptor-associated Gephyrin Splice Variants

Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRbeta subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5-40 amino acids, are subject to alternative splicing (C1-C7, C4'-C6'). Since one of the cassettes, C5', has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRbeta loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6', already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5' was coisolated mainly from liver. Notably C5'-containing gephyrin bound to the GlyRbeta loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.

A novel 9-bp insertion in the GJB1 gene causing a mild form of X-linked CMT with late onset

X-linked Charcot-Marie-Tooth disease is the second most common variant of CMT. CMTX1 is caused by mutations in the GJB1 gene encoding for connexin 32. We describe an Italian family with an intermediate CMTX phenotype with late onset. Mutation screening of the GJB1 gene revealed a 9-bp duplication leading to the insertion of three aminoacids (Thr-Val-Phe) between the end of the second extracellular domain and the beginning of the fourth transmembrane domain. This is the third in-frame insertion in the GJB1 gene identified so far and, like the previous ones, it consists in the duplication of the flanking sequence which is repeated in tandem in the wild-type gene.

Evolutionarily Conserved Allosteric Network in the Cys Loop Family of Ligand-gated Ion Channels Revealed by Statistical Covariance Analyses

The Cys loop family of ligand-gated ion channels mediate fast synaptic transmission for communication between neurons. They are allosteric proteins, in which binding of a neurotransmitter to its binding site in the extracellular amino-terminal domain triggers structural changes in distant transmembrane domains to open a channel for ion flow. Although the locations of binding site and channel gating machinery are well defined, the structural basis of the activation pathway coupling binding and channel opening remains to be determined. In this paper, by analyzing amino acid covariance in a multiple sequence alignment, we have identified an energetically interconnected network in the Cys loop family of ligand-gated ion channels. Statistical coupling and correlated mutational analyses along with clustering revealed a highly coupled cluster. Mapping the positions in the cluster onto a three-dimensional structural model demonstrated that these highly coupled positions form an interconnected network linking experimentally identified binding domains through the coupling region to the gating machinery. In addition, these highly coupled positions are also condensed in the transmembrane domains, which are a recent focus for the sites of action of many allosteric modulators. Thus, our results revealed a genetically interconnected network that potentially plays an important role in the allosteric activation and modulation of the Cys loop family of ligand-gated ion channels.

Studies on the Insolubility of a Transmembrane Peptide from Signal Peptide Peptidase

We have undertaken fundamental studies on the solubility properties of a peptide derived from the fourth transmembrane (TM) domain of signal peptide peptidase, a 7-TM intramembrane-cleaving protease. We have found that by disfavoring secondary structure formation we are able to greatly improve the solubility, handling, and purification properties of this peptide. Our findings suggest that preventing secondary structure formation by reversible modification of the polypeptide backbone of hydrophobic transmembrane peptides may be a useful strategy for the total chemical protein synthesis of integral membrane proteins.

Ion channels and epilepsy

In order for cells to retain their integrity to water and yet permeate charged ions, the phospholipid cell membrane contains transmembrane proteins that allow the passage of specific ions from the interior of the cell to outside, and vice versa. There is a huge diversity of these ion channels. Some are tissue-specific; others are widely distributed throughout the body. They contribute to the maintenance of the negative resting membrane potential inside cells. Unsurprisingly, these membrane channels are integral to the processes of electrical signalling and excitation that are central to the functioning of the nervous system. Figure 1a shows a generic ion channel. Figure 1. ( A ) Diagrammatic representation of a voltage-gated ion channel α subunit. This could be a sodium or calcium channel. Note the four homologous repeats (I–IV), each with six transmembrane domains (1–6). The fourth transmembrane domain (4) has positively charged segments and acts as the voltage sensor. ( B ) Diagrammatic representation of an assembled calcium channel with auxiliary (β, α2δ and γ) subunits. The four homologous repeats (I–IV) are marked on the α1 subunit, and come together to make the channel pore. The assembly of sodium channels is similar, but only has auxiliary β subunits. The past 15 years have seen rapid expansion in the discovery of disease-causing mutations in genes encoding ion channel proteins. These manifest as neurological, cardiac, renal and respiratory disorders, the most common being cystic fibrosis. This disparate collection of syndromes is now referred to as the channelopathies, a descriptive term referring to the common underlying pathophysiology of ion channel dysfunction. Ion channels themselves are divided into two broad categories, depending on their mode of activation. Voltage-gated channels are controlled by changes in membrane potential, ligand-gated channels by ligand binding (Table 1). View this table: Table 1 Ion channels implicated in epilepsy Epilepsy affects up to … Address correspondence to Dr T.D. Graves, Department of Molecular Neurosciences, Institute of Neurology, University College London, Queen Square, London WC1N 3BG. email: t.graves{at}ion.ucl.ac.uk

Tryptophan scanning mutagenesis of the HERG K+ channel: the S4 domain is loosely packed and likely to be lipid exposed

Inherited mutations or drug-induced block of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death. The fourth transmembrane domain (S4) of these channels contains multiple positive charges that move across the membrane electric field in response to changes in transmembrane voltage. In HERG K+ channels, the movement of the S4 domain across the transmembrane electric field is particularly slow. To examine the basis of the slow movement of the HERG S4 domain and specifically to probe the relationship between the S4 domain with the lipid bilayer and rest of the channel protein, we individually mutated each of the S4 amino acids in HERG (L524-L539) to tryptophan, and characterized the activation and deactivation properties of the mutant channels in Xenopus oocytes, using two-electrode voltage-clamp methods. Tryptophan has a large bulky hydrophobic sidechain and so should be tolerated at positions that interact with lipid, but not at positions involved in close protein-protein interactions. Significantly, we found that all S4 tryptophan mutants were functional. These data indicate that the S4 domain is loosely packed within the rest of the voltage sensor domain and is likely to be lipid exposed. Further, we identified residues K525, R528 and K538 as being the most important for slow activation of the channels.

Subtype-dependence of N-methyl-d-aspartate receptor modulation by pregnenolone sulfate

N-methyl-d-aspartate receptors play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits. The subunit composition determines the biophysical and pharmacological properties of the N-methyl-d-aspartate receptor channel complex. In this study, we report that responses mediated by recombinant rat N-methyl-d-aspartate receptors expressed in human embryonic kidney HEK293 cells are differentially affected by naturally occurring neurosteroid pregnenolone sulfate. We show that responses induced by 1mM glutamate in NR1-1a/NR2A and NR1-1a/NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR1-1a/NR2C and NR1-1a/NR2D receptors with no differences in the concentration of pregnenolone sulfate that produced 50% potentiation. In addition to potentiation, pregnenolone sulfate also has an inhibitory effect at recombinant N-methyl-d-aspartate receptors, with values of the concentration of pregnenolone sulfate that produces 50% inhibition of NR1/NR2D=NR1/NR2C<NR1/NR2B<NR1/NR2A. In addition, we show that the structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit is critical for both the potentiating and inhibitory effects of pregnenolone sulfate. The modulatory effects of pregnenolone sulfate are consistent with a model in which this neurosteroid acts at two distinct binding sites on the N-methyl-d-aspartate receptor. These data provide insight into the mechanisms by which pregnenolone sulfate and related sulfated neurosteroids modulate activity of N-methyl-d-aspartate receptor channels.

20-oxo-5beta-pregnan-3alpha-yl sulfate is a use-dependent NMDA receptor inhibitor.

NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) action at NMDA receptors. 3alpha5betaS was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3alpha5betaS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3alpha5betaS in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3alpha5betaS at NMDA receptors. In accordance with the model, 3alpha5betaS was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3alpha5betaS is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.

Sphingosine 1-phosphate analogue recognition and selectivity at S1P4 within the endothelial differentiation gene family of receptors

Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P4 G-protein-coupled receptor. Similarly to reports on the S1P1 receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P1 receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp186 at position 4.64 in the fourth transmembrane domain interacts by a cation-pi interaction with the ammonium group of S1P. Lys204 at position 5.38 forms an ion pair with the S1P. The S1P4 and S1P1 receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure-activity relationships. In particular, the binding pocket of mS1P4 (mouse S1P4) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P1 (human S1P4) places the ammonium recognition site 2 A (1 A=0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site.

Defining a Minimal Motif Required to Prevent Connexin Oligomerization in the Endoplasmic Reticulum

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.

Tryptophan scanning mutagenesis suggests that the voltage sensor in HERG has a peripheral location

HERG encodes the α-subunit of the delayed rectifier K+ channel that contributes to cardiac repolarization. The fourth trans-membrane domain (S4) of HERG, contains multiple positive charges and is the voltage sensor for activation. However, the mechanisms by which the S4 segment senses changes in voltage and moves in response to those voltage changes remains controversial. The paddle model of MacKinnon suggests that S4 lies in a lipid environment and moves freely across the membrane, whereas the focused field model predicts that the charged residues in S4 make specific protein-protein interactions and are therefore protected from the hydrophobic membrane environment.

Roles for Nicotinic Acetylcholine Receptor Subunit Large Cytoplasmic Loop Sequences in Receptor Expression and Function

To evaluate possible physiological roles of the large cytoplasmic loops (C2) and neighboring transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits, we generated novel fusion constructs in which human nAChR alpha4, beta2, or beta4 subunit C2 or C2 and neighboring sequences were replaced by corresponding sequences from the mouse serotonin type 3A (5-HT3A) receptor subunit. Following stable expression in human SH-EP1 cells, we found that extensive sequence substitutions involving third and fourth transmembrane domains and neighboring "proximal" C2 sequences (e.g., beta2 H322-V335 and V449-R460) did not allow functional expression of nAChR containing chimeric subunits. However, expression of functional nAChR was achieved containing wild-type alpha4 subunits and chimeric beta2 (beta2chi) subunits whose "nested" C2 domain sequences K336-S448 were replaced with the corresponding 5-HT3A subunit sequences. Whereas these findings suggested indispensable roles for M3/M4 transmembrane and/or proximal C2 sequences in alpha4beta2-nAChR function, nested C2 sequences in the beta2 subunit are not essential for functional receptor expression. Ligand-binding analyses also revealed only subtle differences in pharmacological profiles of alpha4beta2-nAChR compared with alpha4beta2chi-nAChR. Nevertheless, there was heightened emergence of agonist-mediated self-inhibition of alpha4beta2chi function, greater sensitivity to functional blockade by a number of antagonists, and faster and more complete acute desensitization of alpha4beta2chi-nAChR than for alpha4beta2-nAChR. These studies are consistent with unexpected roles of nested C2 sequences in nAChR function.

Identification and characterization of a novel human APH-1b splice variant lacking exon 4

APH-1 is one of the four essential components of the presenilin-γ-secretase complex and has two human homologs, APH-1a, and APH-1b, both of which are seven-pass membrane proteins. Here, we identified a novel splice variant of human APH-1b. This variant lacks exon 4, which encodes the entire fourth transmembrane domain. The mRNA expression of this variant was detected in most tissues at low levels. In transiently transfected cells, protein expression of the APH-1b variant was much lower than that of the wild-type. Furthermore, exogenous expression of the APH-1-interacting protein, nicastrin, significantly increased the variant protein levels. These data suggest that the APH-1b variant protein is destabilized, and implies that the fourth transmembrane domain plays an important role in the protein stability and function of APH-1.

Palmitoylation of claudins is required for efficient tight-junction localization

Palmitoylation of integral membrane proteins can affect intracellular trafficking, protein-protein interactions and protein stability. The goal of the present study was to determine whether claudins, transmembrane-barrier-forming proteins of the tight junction, are palmitoylated and whether this modification has functional implications for the tight-junction barrier. Claudin-14, like other members of the claudin family, contains membrane-proximal cysteines following both the second and the fourth transmembrane domains, which we speculated could be modified by S-acylation with palmitic acid. We observed that [(3)H]-palmitic acid was incorporated into claudin-14 expressed by transfection in both cultured epithelial cells and fibroblasts. Mutation of cysteines to serines following either the second or the fourth transmembrane segments decreased the incorporation of [(3)H]-palmitic acid, and mutation of all four cysteines eliminated palmitoylation. We previously reported that expression of claudin-14 in epithelial monolayers results in a fivefold increase in electrical resistance. By contrast, expression of the mutant claudin-14 resulted in smaller increases in resistance. The mutants localized less well to tight junctions and were also found in lysosomes, suggesting an alteration in trafficking or stability. However, we observed no change in protein half-life and only a small shift in fractionation out of caveolin-enriched detergent-resistant membranes. Although less well localized to the tight junction, palmitoylation-deficient claudin-14 was still concentrated at sites of cell-cell contact and was competent to assemble into freeze-fracture strands when expressed in fibroblasts. These results demonstrate that palmitoylation of claudin-14 is required for efficient localization into tight junctions but not stability or strand assembly. Decreased ability of the mutants to alter resistance is probably the result of their less efficient localization into the barrier.