HCN channels are thought to be structurally similar to Kv channels, but show much lower selectivity for K+. The ∼3.3 Å selectivity filter of K+ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L478C479IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K+ channels, we also studied C479T channels. Based on permeability ratios (PX/PK), C479T HCN4 channels (K+(1)>Rb+(0.85)>Cs+(0.59)>Li+(0.50)≥Na+(0.49)) were less selective than WT rabbit HCN4 (K+(1)>Rb+(0.48)>Cs+(0.31)≥Na+(0.29)>Li+(0.03)), indicating that the TIGYG sequence is insufficient to confer K+ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K+ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K+ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K+ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K+ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K+ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K+ channels originate from differences outside the P-loop region.
Cardiac inward rectifier K+ currents (IK1) play an important role in maintaining resting membrane potential and contribute to late phase repolarization. Members of the Kir2.x channel family appear to encode for IK1. The purpose of this study was to determine the molecular composition of cardiac IK1 in rabbit ventricle. Western blots revealed that Kir2.1 and Kir2.2, but not Kir2.3, are expressed in rabbit ventricle. Culturing rabbit myocytes resulted in an approximately 50% reduction of IK1 density after 48 or 72 h in culture which was associated with an 80% reduction in Kir2.1, but no change in Kir2.2, protein expression. Dominant-negative (DN) constructs of Kir2.1, Kir2.2 and Kir2.3 were generated and tested in tsA201 cells. Adenovirus-mediated over-expression of Kir2.1dn, Kir2.2dn or Kir2.1dn plus Kir2.2dn in cultured rabbit ventricular myocytes reduced IK1 density equally by 70% 72 h post-infection, while AdKir2.3dn had no effect, compared to green fluorescent protein (GFP)-infected myocytes. Previous studies indicate that the [Ba2+] required for half-maximum block (IC50) differs significantly between Kir2.1, Kir2.2 and Kir2.3 channels. The dependence of IK1 on [Ba2+] revealed a single binding isotherm which did not change with time in culture. The IC50 for block of IK1 was also unaffected by expression of the different DN genes after 72 h in culture. Taken together, these results demonstrate functional expression of Kir2.1 and Kir2.2 in rabbit ventricular myocytes and suggest that macroscopic IK1 is predominantly composed of Kir2.1 and Kir2.2 heterotetramers.
Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH metabolism is abnormal, and many biochemical and functional alterations mirror hypothyroidism. Although TH therapy has been advocated for treating heart disease, a clear benefit of TH has yet to be established, possibly because of peripheral actions of TH. To assess the potential efficacy of TH in treating heart disease, type 2 deiodinase (D2), which converts the prohormone thyroxine to active triiodothyronine (T3), was expressed transiently in mouse hearts by using the tetracycline transactivator system. Increased cardiac D2 activity led to elevated cardiac T3 levels and to enhanced myocardial contractility, accompanied by increased Ca(2+) transients and sarcoplasmic reticulum (SR) Ca(2+) uptake. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) 2a expression as well as decreased Na(+)/Ca(2+) exchanger, beta-myosin heavy chain, and sarcolipin (SLN) expression. In pressure overload, targeted increases in D2 activity could not block hypertrophy but could completely prevent impaired contractility and SR Ca(2+) cycling as well as altered expression patterns of SERCA2a, SLN, and other markers of pathological hypertrophy. Our results establish that elevated D2 activity in the heart increases T3 levels and enhances cardiac contractile function while preventing deterioration of cardiac function and altered gene expression after pressure overload.
Ion channel conductance can be influenced by electrostatic effects originating from fixed “surface” charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K+ activities (4.6–293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K+ activities. K+ ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC4(5). The dependence of γ on the K+ activity (aK) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as aK approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-d-glucamine (NMG+), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the γ–aK relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic γ–aK relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge.
Introduction: Voltage sensor mutations may cause hypokalemic periodic paralysis. We report a sodium channel voltage sensor mutation producing dilated cardiomyopathy and a spectrum of arrhythmias wi...
Abstract Background Cystic Fibrosis causing mutations in the gene CFTR , reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. Methods Small molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. Results We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. Conclusion Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.
Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg78, Ser159, or Thr182 to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist l-[3H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser157 and Ser160) or a relatively modest effect (Ser181) on ligand affinity compared with the Arg78, Ser159, and Thr182 mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog l-serineO-phosphate (l-SOP) was “docked” into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser159 and Thr182 of mGluR4 form hydrogen bonds with the α-carboxyl and α-amino groups on l-SOP, respectively, whereas Arg78 forms an electrostatic interaction with the acidic side chains of l-SOP or glutamate. The conservation of Arg78, Ser159, and Thr182 in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors. Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg78, Ser159, or Thr182 to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist l-[3H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser157 and Ser160) or a relatively modest effect (Ser181) on ligand affinity compared with the Arg78, Ser159, and Thr182 mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog l-serineO-phosphate (l-SOP) was “docked” into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser159 and Thr182 of mGluR4 form hydrogen bonds with the α-carboxyl and α-amino groups on l-SOP, respectively, whereas Arg78 forms an electrostatic interaction with the acidic side chains of l-SOP or glutamate. The conservation of Arg78, Ser159, and Thr182 in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors. metabotropic glutamate receptors l-amino-4-phosphonobutyric acid serineO-phosphate γ-aminobutyric acid, type B amino-terminal domain leucine/isoleucine/valine-binding protein human embryonic kidney polymerase chain reaction (2S,3S,4S)-CCG/(2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (RS)-α-cyclopropyl-4-phosphonophenylglycine phosphate-buffered saline Metabotropic glutamate receptors (mGluRs)1 are a family of eight G-protein-coupled receptors that are expressed throughout the central nervous system and in sensory cells of the retina and tongue. The mGluR family has been divided into three subgroups based on sequence homology, pharmacology, and signal transduction properties; in cell lines, group I mGluRs couple to phosphoinositide turnover, whereas group II and III receptors couple to the inhibition of forskolin-stimulated cAMP via Gi/Go proteins (1Pin J.-P. Duvoisin R. Neuropharmacology. 1995; 34: 1-26Crossref PubMed Scopus (1233) Google Scholar, 2Gomeza J. Mary S. Brabet I. Parmentier M.-L. Restituito S. Bockaert J. Pin J.-P. Mol. Pharmacol. 1996; 50: 923-930PubMed Google Scholar). mGluR4 together with mGluR6, mGluR7, and mGluR8 constitute the group III subclass of mGluRs that are selectively sensitive to the phosphono derivative of l-glutamate,l-amino-4-phosphonobutyric acid (l-AP4), and the endogenous amino acid l-serine O-phosphate (l-SOP). The group III mGluRs are important regulators of synaptic transmission in the central nervous system. Electrophysiological experiments have shown that activation of l-AP4-sensitive receptors causes a suppression of synaptic transmission by inhibiting neurotransmitter release from nerve terminals (3Macek T.A. Winder D.G. Gereau IV, R.W. Ladd L.O. Conn J.P. J. Neurosci. 1996; 76: 3798-3806Google Scholar), and immunocytochemical studies have confirmed that group III mGluRs are localized presynaptically (4Risso Bradley S. Standaert D.G. Rhodes K.J. Rees H.D. Testa C.M. Levey A.I. Conn P.J. J. Comp. Neurol. 1999; 407: 33-46Crossref PubMed Scopus (133) Google Scholar, 5Shigemoto R. Kinoshita A. Wada E. Nomura S. Ohishi H. Takada M. Flor P.J. Neki A. Abe T. Nakanishi S. Mizuno N. J. Neurosci. 1997; 17: 7503-7522Crossref PubMed Google Scholar, 6Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The characterization of mutant mice lacking the mGluR4 subtype of mGluR has provided additional insight into the function of this receptor in the nervous system. For example, observations from electrophysiological analyses demonstrating impaired presynaptic functions in the mutant mice led to the suggestion that this receptor may be required for sustaining synaptic transmission during periods of high-frequency neurotransmission (7Pekhletski R. Gerlai R. Overstreet L. Huang X.-P. Agoypan N. Slater N.T. Roder J.C. Hampson D.R. J. Neurosci. 1996; 16: 6364-6373Crossref PubMed Google Scholar). Behavioral studies on mGluR4 mutant mice have shown that this receptor plays a role in motor and spatial learning (7Pekhletski R. Gerlai R. Overstreet L. Huang X.-P. Agoypan N. Slater N.T. Roder J.C. Hampson D.R. J. Neurosci. 1996; 16: 6364-6373Crossref PubMed Google Scholar,8Gerlai R. Roder J.C. Hampson D.R. Behav. Neurosci. 1998; 112: 1-8Crossref Scopus (54) Google Scholar). The potential use of group III mGluR ligands as therapeutic agents in epilepsy and neurodegenerative disorders has provided a persuasive argument for conducting more detailed structural analyses of this class of neurotransmitter receptors (9Nicoletti F. Bruno V. Copani A. Casabona G. Knopfel T. Trends Neurosci. 1996; 19: 267-272Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 10Thomsen C. Dalby N.O. Neuropharmacology. 1998; 37: 1465-1473Crossref PubMed Scopus (65) Google Scholar). The amino acid sequences of the mGluRs are homologous to the periplasmic amino acid-binding proteins in bacteria (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar), the calcium-sensing receptor of the parathyroid gland (12Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2351) Google Scholar, 13Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (343) Google Scholar), the GABAB receptors (14Kaupmann K. Huggel K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froesti W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (874) Google Scholar, 15Kuner R. Kohr G. Grunewald S. Eisenhardt G. Bach A. Kornau H.-C. Science. 1999; 283: 74-77Crossref PubMed Scopus (493) Google Scholar, 16Ng G.Y.K. Clark J. Coulombe N. Ethier N. Hebert T.E. Sullivan R. Kargman S. Chateauneuf A. Tsukamoto N. McDonald T. Whiting P. Mezey E. Johnson M.P. Liu Q. Kolakowski Jr., L.F. Evans J.F. Bonner T.I. O'Neill G.P. J. Biol. Chem. 1999; 274: 7607-7610Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), a group of mammalian pheromone receptors (17Matsunami H. Buck L.B. Cell. 1997; 90: 775-788Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar), and a class of taste receptors expressed in lingual tissue (18Moon M.A. Alder E. Lindemeier J. Battey J.F. Ryba N.J.P. Zucker C.S. Cell. 1999; 96: 541-551Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar). The basic structural domains of mGluRs include a large extracellular amino-terminal domain (ATD), seven putative transmembrane domains, and an intracellular carboxyl terminus. The homology of the ATDs of mGluRs to the leucine/isoleucine/valine-binding protein (LIVBP) and other bacterial periplasmic binding proteins that mediate the transport of amino acids in prokaryotes is fortuitous because the mGluRs appear to possess a similar three-dimensional fold and the crystal structures of the bacterial proteins are known (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar). Data obtained from experiments on chimeric constructs of the ATD of human mGluR4 with the transmembrane domains and carboxyl-terminal regions of mGluR1b (19Tones M.A. Bendali H. Flor P.J. Knopfel T. Kuhn R. Neuroreport. 1995; 7: 117-120Crossref PubMed Google Scholar) and constructs containing various segments of the ATD of rat mGluR2 and the transmembrane domain and carboxyl terminus of mGluR1a (20Takahashi K. Tsuchida K. Tanabe Y. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 19341-19345Abstract Full Text PDF PubMed Google Scholar) indicated that pharmacological selectivity is conferred by residues located in the ATDs of mGluRs. More recent studies demonstrating that the ATDs of mGluR1 (21Okamoto T. Sekiyama N. Otsu M. Shimada Y. Sato A. Nakanishi S. Jingami H. J. Biol. Chem. 1998; 273: 13089-13096Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and mGluR4 (22Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) can be expressed as soluble proteins that are secreted from transfected cells and that retain ligand-binding capabilities have corroborated the concept that the primary determinants of ligand binding to mGluRs are contained within the ATDs. In this study, we have employed molecular modeling in conjunction with site-directed mutagenesis to probe the ligand-binding pocket of mGluR4. Our results indicate that three conserved amino acids present in the ATDs may be key determinants of ligand binding to all members of the mGluR family. The three-dimensional structure of the proposed ligand-binding domain of rat mGluR4 was formulated by homology modeling using the experimentally determined structure of the closed form of LIVBP from Escherichia coli and the strategy outlined by Blundell et al. (23Blundell T.L. Sibanda B.L. Sternberg M.J.E. Thornton J.M. Nature. 1987; 326: 347-352Crossref PubMed Scopus (589) Google Scholar). The atomic coordinates for the closed form of LIVBP with leucine in the binding pocket were kindly provided by Dr. F. A. Quiocho (Baylor College of Medicine). The QUANTA program (Version 97, MSI Corp.) and the SYBYL program (Version 6.4, Tripos Associates) were used to view the model that encompassed the region from Gly47 to Lys490 in the ATD of mGluR4. The sequence alignment used in the mGluR4 model has been described previously (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar). Backbone atom coordinates were assigned the corresponding residue coordinates from the crystal structure of LIVBP, and side chain atom coordinates were based on maximal side chain atom fitting to the LIVBP structure. Regions with insertions or deletions were modeled using known substructures identified by loop-searching techniques; regions 1–46, 125–149, 353–401, and 426–439, which are absent in LIVBP, were not included in the model. The l-SOP molecule was docked into the binding site of mGluR4 in an orientation that corresponds to that observed for leucine binding to LIVBP. The model was energy-optimized using a restrained energy minimization with additional constraints applied to the backbone regions based on the x-ray structure of LIVBP using the CHARMm force field. A steepest descent followed by a conjugate gradient method were used for energy minimization until the energy change per cycle was <0.0001 kcal/mol. For the expression of wild-type mGluR4a in human embryonic kidney cells (HEK-293-TSA-201), the BglII-EcoRI fragment of mGluR4a in the pBluescript SK− phagemid (m4aSK−) (24Tanabe Y. Masu M. Ishii T. Shigemoto R. Nakanishi S. Neuron. 1992; 8: 169-179Abstract Full Text PDF PubMed Scopus (889) Google Scholar) was subcloned into the pcDNA3 mammalian expression vector (Invitrogen, San Diego, CA) at the BamHI and EcoRI sites. For the construction of c-Myc-tagged mGluR4a, the mGluR4a-pcDNA3 plasmid was cut with XhoI, and the larger fragment containing pcDNA3 backbone was ligated to itself (the 5′-mGluR4a-pcDNA3 plasmid). The primersBstEII-c-Myc (5′-GT CAC GAA CAA AAG CTT ATT TCT GAA GAA GAC TTG GAT CCA G) and rev-BstEII-c-Myc (5′-GTG ACC TGG ATC CAA GTC TTC TTC AGA AAT AAG CTT TTG TTC) were phosphorylated, annealed to each other, and cloned into the 5′-mGluR4a-pcDNA3 plasmid at the dephosphorylated BstEII site to produce 5′-mGluR4a-c-Myc-pcDNA3. The 931-base pairNdeI-XhoI fragment from 5′-mGluR4a-c-Myc-pcDNA3 and a 3335-base pairXhoI-NotI fragment of mGluR4a-pcDNA3 were subcloned into pcDNA3 at NdeI-NotI sites using a three-piece ligation. The c-Myc-tagged mutants were also constructed in this manner using the corresponding mutants. For the generation of the S157A, S160A, and S181A mutants, the sequences flanking the point of mutation were amplified in two separate PCRs on the rat mGluR4a-pcDNA3 expression plasmid. For all other mutants, the mGluR4a cDNA in pBluescript SK−(Stratagene) was used as the template. One of four primers used in the generation of each mutant contained the desired mutation. An adjacent primer was phosphorylated prior to PCR, and the two PCR products were ligated to each other and reamplified using the two most distant primers (the 5′-primer from the first PCR and the 3′-primer from the second PCR). The resulting products were cut with the appropriate restriction enzymes and subcloned in place of the corresponding wild-type fragment. All expression constructs were assembled in the pcDNA3 mammalian expression vector for transient transfection in HEK cells. In all cases, the orientation of the inserts and the integrity of subcloning sites were checked by restriction analysis where applicable, and the PCR-amplified regions were sequenced to confirm the mutations and to ensure that no other changes were introduced. A cassette mutagenesis method was used to construct the S159A mutation. A 1.79-kilobase KpnI fragment of mGluR4a containing Ser159 was subcloned into the pBluescript SK− vector and transformed into CJ236 bacteria. A mutagenic oligonucleotide (5′-GGA GCT TCA GGG GCC TCC GTC TCG ATC A-3′) was annealed to the template and used to make double-stranded mutant DNA with T4 DNA polymerase and T4 ligase. The double-stranded mutant DNA was transformed into DH5α cells (Life Technologies, Inc.), and rapid screening of the colonies was carried out using theSacI restriction enzyme; the DNA from a positive colony was sequenced to confirm the presence of the S159A mutation and the absence of any additional base pair changes. The mutated cassette was then excised from pBluescript SK− and ligated back in the correct orientation in the mGluR4a cDNA in pcDNA3. HEK cells were cultured in modified Eagle's medium with 6% fetal bovine serum and antibiotics. Transient transfections were conducted using the protocol described previously (22Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar); all experiments were conducted on cells or membranes collected 48 h after transfection. The membrane preparation procedure and the l-[3H]AP4 binding assay were carried out as described by Eriksen and Thomsen (25Eriksen L. Thomsen C. Br. J. Pharmacol. 1995; 116: 3279-3287Crossref PubMed Scopus (46) Google Scholar), except that 300 μml-SOP was used to define nonspecific binding. Bound and free radioligands were separated by centrifugation. For competition experiments, 30 nml-[3H]AP4 was used. The data were analyzed using GraphPAD Prism software. l-[3H]AP4 (specific activity, 54 Ci/mmol), l-SOP, sodiuml-glutamate, (2S,3S,4S)-CCG/(2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (l-CCG-1), and (RS)-α-cyclopropyl-4-phosphonophenylglycine) (CPPG) were purchased from Tocris (Bristol, United Kingdom) The procedures for immunoblotting were carried out as described by Pickering et al. (26Pickering D.S. Taverna F.A. Salter M.W. Hampson D.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12090-12094Crossref PubMed Scopus (71) Google Scholar). Electrophoresis samples containing 100 mmdithiothreitol were incubated at 37 °C for 15 min prior to gel electrophoresis. Antibodies raised in rabbits against the carboxyl terminus of mGluR4a were generated as described by Risso Bradleyet al. (4Risso Bradley S. Standaert D.G. Rhodes K.J. Rees H.D. Testa C.M. Levey A.I. Conn P.J. J. Comp. Neurol. 1999; 407: 33-46Crossref PubMed Scopus (133) Google Scholar) and Petralia et al. (27Petralia R.S. Wang Y.-X. Niedzielski A.S. Wenthold R.J. Neuroscience. 1996; 71: 949-976Crossref PubMed Scopus (515) Google Scholar). For immunocytochemical analyses, HEK cells were washed with phosphate-buffered saline (PBS) for 2 × 2 min at 48 h post-transfection and fixed with PBS containing 4% paraformaldehyde and 4% sucrose for 10 min at 25 °C. The cells were air-dried for 15 min and then incubated in 10% bovine serum albumin in PBS for 30 min at 25 °C. The cells were subsequently incubated for 1 h at 25 °C with either the anti-mGluR4a antibody or with anti-c-Myc mouse monoclonal IgG1 (Upstate Biotechnology, Inc.) diluted to a final concentration of 0.15 μg/ml in 3% bovine serum albumin in PBS. The primary antibody was then removed, and the cells were washed 5 × 5 min with PBS. After washing, the cells were incubated for 60 min at 25 °C with biotin-conjugated anti-mouse IgG (Sigma, B 0529) diluted to a final concentration of 2.75 μg/ml in 3% bovine serum albumin in PBS. After incubation, the cells were washed 5 × 5 min with PBS and treated with fluorescein isothiocyanate-conjugated ExtrAvidin (Sigma, E 2761) diluted to a final concentration of 5 μg/ml in 3% bovine serum albumin in PBS for 60 min at 25 °C in the dark; the cells were washed 4 × 5 min with PBS, mounted with 50% glycerol solution in PBS, and photographed with a Zeiss Axiovert 135 TV microscope equipped with a 485-nm excitation and 530-nm emission filter at a magnification of ×400. HEK cells were subcultured onto six-wells plates 1 day before transfection at 50% confluency. The cells were cotransfected with 4 μg of mGluR4a cDNA or mutant cDNAs and 4 μg of Gqi9 cDNA in the pcDNA1 vector (28Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (603) Google Scholar). At 24 h post-transfection, the cells were plated onto 35-mm dishes (Nunc) fitted with glass coverslips (Bellco Glass, Inc.) previously coated overnight at 37 °C with poly-l-ornithine (0.01%, M r 40,000; Sigma) to increase adhesion of the cells. At 48 h post-transfection, the cells were washed 3 × 5 min at 37 °C in wash buffer (135 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.9 mm MgCl2, and 10 mm HEPES, pH 7.4), and then loaded for 45 min at 37 °C with 6 μm fura-2 acetoxymethyl ester (Molecular Probes, Inc.) dissolved in wash buffer. After loading, the cells were washed 3 × 10 min with wash buffer prior to recording. Fluorescence recordings were made on single cells using a dual excitation imaging system (Universal Imaging Corp.) equipped with a Zeiss Axiovert 135 microscope. The ATD of mGluR4 extends from the amino terminus to the first putative transmembrane domain and encompasses the initial 66 kDa of the receptor protein (Fig.1). The molecular model of the ATD of mGluR4 retains the salient characteristics of the bacterial periplasmic binding proteins. These include two domains of similar shape connected by a hinge region made up of three interdomain crossover segments (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar,29Sack J.S. Saper M.A. Quiocho F.A. J. Mol. Biol. 1989; 206: 171-191Crossref PubMed Scopus (223) Google Scholar). The large insertions at amino acids 1–46, 125–149, 353–401, and 426–439 that were not included in the model are all well separated from the proposed ligand-binding site located in a cavity formed between the two domains. This site is analogous to the leucine-binding site found in LIVBP. In this cavity, the agonist l-SOP is held in place by hydrogen bond interactions with both main chain and side chain atoms and complementary ionic interactions with charged residues. With the exception of hydrogen bonds between the ligand and the peptide backbone of the binding domain, these interactions can be disrupted by substituting the natural amino acids with alanine. Thus, a series of mutations were made at selected residues that were anticipated to interact directly with the ligand (Arg78, Ser159, and Thr182) and at amino acids that may be indirectly involved in binding (Ser157 and Ser181). The model predicted that Ser160 lies outside of the binding pocket, and therefore mutation of this residue to alanine was not likely to affect ligand binding. To determine whether any of the point mutations affected protein expression, immunoblots of cells transiently transfected with mGluR4a or with the R78A, S157A, S159A, S160A, S181A, or T182A mutant were probed with an antibody raised against the carboxyl terminus of mGluR4a. Labeled bands with relative molecular masses of ∼96 and 100 kDa, which likely correspond to the non-glycosylated and glycosylated forms of mGluR4, respectively, were observed in samples of wild-type and c-Myc-tagged mGluR4a and in all of the mutants (Fig. 2). Higher molecular mass dimers of mGluR4 were also present as previously reported in mouse cerebellum (7Pekhletski R. Gerlai R. Overstreet L. Huang X.-P. Agoypan N. Slater N.T. Roder J.C. Hampson D.R. J. Neurosci. 1996; 16: 6364-6373Crossref PubMed Google Scholar). The R78A mutant also showed an additional immunoreactive band at ∼90 kDa; the nature of this band is not known. Nevertheless, the intensity of the monomer bands at 96 and 100 kDa was similar to that of the wild-type receptor in all mutants including R78A, demonstrating that none of the point mutations produced any substantial alterations in the level of protein expression. The similarity in the expression levels of wild-type mGluR4a and the S157A, S160A, and S181A mutants was also indicated by the similarB max values in the radioligand binding experiments (see below). Saturation analyses ofl-[3H]AP4 binding to membranes prepared from HEK cells transfected with the wild-type mGluR4a expression plasmid showed a dissociation constant (K D ) and maximum number of binding sites (B max) of 504 nm and 8.6 pmol/mg, respectively (Fig.3 A and TableI). The dissociation constant for mGluR4a expressed in HEK cells was similar to that reported previously for mGluR4a expressed in hamster kidney cells (K D = 441 nm) (25Eriksen L. Thomsen C. Br. J. Pharmacol. 1995; 116: 3279-3287Crossref PubMed Scopus (46) Google Scholar) and in insect Sf9 cells (K D = 480 nm) (30Thomsen C. Pekhletski R. Haldeman B. Gilbert T.A. O'Hara P.J. Hampson D.R. Neuropharmacology. 1997; 36: 21-30Crossref PubMed Scopus (44) Google Scholar). A modified expression vector was also constructed in which a c-Myc epitope tag was inserted immediately downstream of the proposed signal peptide (Fig.1). The insertion of the c-Myc tag at this position was done (a) to provide an extracellular antibody epitope to facilitate immunocytochemical labeling (see below) and (b) to ensure that the tag would not be cleaved by signal peptidases. c-Myc-tagged mGluR4a displayed K D andB max values of 404 nm and 8.7 pmol/mg, respectively (Fig. 3 B and Table I); neither value was significantly different (p > 0.05, one-way analysis of variance and Dunnett's multiple comparison test) from that of the untagged receptor, indicating that the insertion of the epitope at this site did not affect ligand affinity or the level of expression of mGluR4a.Table IAffinity constants and maximal binding capacities from l-[ 3 H]AP4 saturation binding analyses conducted on wild-type mGluR4a, c-Myc-tagged mGluR4a, and mutant receptorsmGluR4aK DB maxnmpmol/mgWT1-aWild-type.504 ± 998.6 ± 2.9c-Myc-WT404 ± 648.7 ± 1.3S157A683 ± 526.3 ± 1.0S160A470 ± 725.0 ± 1.6S181A570 ± 524.2 ± 1.2Each value is the mean ± S.E. of three to four experiments conducted in triplicate.1-a Wild-type. Open table in a new tab Each value is the mean ± S.E. of three to four experiments conducted in triplicate. The molecular model of the ATD of mGluR4 suggests that Arg78, Ser159, and Thr182 interact directly with the glutamate ligand. When mutated to alanine, all three residues produced receptors that were nearly devoid of the ability to bind l-[3H]AP4 (Fig.4). The R78A, S159A, and T182A mutants displayed 2 ± 0.8, 5 ± 1, and 4 ± 2% (mean ± S.E. of three experiments) of control (wild-type mGluR4a) binding, respectively. Due to the very low level of binding of the radioligand, it was not possible to obtain estimates of affinities for these two mutants in saturation or competition experiments. To further probe the ligand-binding domain of mGluR4, several additional mutations were made at amino acid residues that were predicted to be in or very near the binding pocket, but not directly involved in ligand binding. Saturation experiments showed that neither the dissociation constants nor the maximum numbers of binding sites of the S157A, S160A, and S181A mutants were significantly different from those of the wild-type receptor (p > 0.05, one-way analysis of variance and Dunnett's multiple comparison test) (Table I). To assess the pharmacological profile of these mutants, competition experiments were conducted using the agonists l-glutamate,l-SOP, and l-CCG-1 and the group III antagonist CPPG (31Toms N.J. Jane D.E. Kemp M.C. Bedingfield J.S. Roberts P.J. Br. J. Pharmacol. 1996; 119: 851-854Crossref PubMed Scopus (77) Google Scholar). The rank order of potency in the S157A, S160A, and S181A mutants was similar to that observed in the wild-type receptor (l-SOP > l-CCG-1 >l-glutamate > CPPG) (Fig.5). The inhibition constants for these drugs with the S157A and S160A mutants were also similar to those seen with the wild-type receptor (Table II). However, the inhibition constants for the S181A mutant were ∼3–5 times higher than those for the wild-type receptor, indicating that this mutation produced a moderate decrease in affinity for the series of compounds tested.Table IIInhibition constants of various drugs forl-[ 3 H]AP4 binding to wild-type mGluR4a and the S157A, S160A, and S181A mutantsmGluR4aIC50l-SOPl-CCG-1l-GlutamateCPPG μmWild-type2.7 ± 0.54.0 ± 1.55.7 ± 0.524 ± 2.7S157A2.2 ± 0.52.3 ± 0.611 ± 3.610.3 ± 4.3S160A3.2 ± 0.53.8 ± 0.34.0 ± 2.621.3 ± 1.1S181A10 ± 216 ± 4.129 ± 5.369 ± 13The concentration of l-[3H]AP4 was 30 nm. Data are the means ± S.E. of three experiments. Open table in a new tab The concentration of l-[3H]AP4 was 30 nm. Data are the means ± S.E. of three experiments. Although the results from the immunoblot experiments indicated that the R78A, S159A, and T182 mutant polypeptides were translated and expressed at levels comparable to those of the wild-type receptor, it is possible that the very low level of ligand binding of the mutants was caused by misfolding and/or lack of cell-surface expression. To investigate this possibility, an immunocytochemical analysis was carried out on the c-Myc-tagged wild-type receptor, the R78A and T182A mutant receptors, and the untagged S159A receptor. Cell-surface expression was assessed by labeling lightly fixed HEK cells (4% paraformaldehyde for 10 min) with the anti-mGluR4a or anti-c-Myc antibody, followed by a biotinylated anti-rabbit or anti-mouse secondary antibody and a fluorescein isothiocyanate-avidin conjugate. Cells expressing c-Myc-tagged wild-type mGluR4a labeled with the anti-mGluR4a antibody and treated with Triton X-100 to permeabilize the cells showed intense labeling in and particularly around the periphery of the cells, whereas similarly transfected cells not treated with Triton X-100 displayed only background labeling (Fig.6, A and B). The absence of specific immunostaining in unpermeabilized transfected cells indicates that the fixation protocol used (without Triton X-100 treatment) did not cause permeabilization of the cells. The immunolabeling pattern observed wit
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