FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2056581302> ?p ?o ?g. }

• W2056581302 endingPage "18392" @default.
• W2056581302 startingPage "18387" @default.
• W2056581302 abstract "Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membrane-spanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligandsN-allylnormetazocine (SKF10047), ditolylguanidine, and (±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (I K). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition inI K, and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 μm. SKF10047 also inhibited I K with a similar EC50 (14 μm). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5′-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism. Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membrane-spanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligandsN-allylnormetazocine (SKF10047), ditolylguanidine, and (±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (I K). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition inI K, and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 μm. SKF10047 also inhibited I K with a similar EC50 (14 μm). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5′-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism. Sigma receptors are widely distributed in neuronal and nonneuronal tissue and are distinguished by their ability to bind a broad range of chemically unrelated ligands, including (+)-opiates, neuroleptic drugs, ditolylguanidine (DTG), 1The abbreviations used are: DTG, ditolylguanidine; I K, voltage-activated potassium current; IAC, iodoazidococaine; G-protein, guanine-nucleotide binding protein; PBS, phosphate-buffered saline; PPHT, (±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin; GDPβS, 5′-O-(2-thiodiphosphate); GTPγS, guanosine 5′-O-(3-thiotriphosphate).and phencyclidine-related compounds (1Su T.-P. Crit. Rev. Neurobiol. 1993; 7: 187-203PubMed Google Scholar, 2Walker J.M. Hohmann A.G. Hemstreet M.K. Martin W.J. Beierlein M. Roth J.S. Patrick S.L. Carroll F.I. Patrick R.L. Stone T.W. Aspects of Synaptic Transmission. Taylor and Francis, Washington, D. C.1993: 91-112Google Scholar, 3Bowen W.D. Stone T.W. Aspects of Synaptic Transmission. Taylor and Francis, Washington, D. C.1993: 113-136Google Scholar). Although the biological activity of ligands suggests that sigma receptors may be involved in behavioral, psychological, and motor functions (1Su T.-P. Crit. Rev. Neurobiol. 1993; 7: 187-203PubMed Google Scholar, 2Walker J.M. Hohmann A.G. Hemstreet M.K. Martin W.J. Beierlein M. Roth J.S. Patrick S.L. Carroll F.I. Patrick R.L. Stone T.W. Aspects of Synaptic Transmission. Taylor and Francis, Washington, D. C.1993: 91-112Google Scholar, 3Bowen W.D. Stone T.W. Aspects of Synaptic Transmission. Taylor and Francis, Washington, D. C.1993: 113-136Google Scholar), the cellular actions of sigma receptors are poorly understood. Recent studies in melanotrophs (4Soriani O. Vaudry H. Mei Y.A. Roman F. Cazin L. J. Pharmacol. Exp. Ther. 1998; 286: 163-171PubMed Google Scholar) and the neurohypophysis (5Wilke R.A. Lupardus P.J. Grandy D.K. Rubinstein M. Low M.J. Jackson M.B. J. Physiol. (Lond.). 1999; 517: 391-406Crossref Scopus (58) Google Scholar) showed that sigma receptor ligands inhibit voltage-activated potassium current (I K), but the signal transduction pathways associated with sigma receptor activation remain unknown. Molecular characterization of sigma receptors has raised intriguing questions about how these receptors generate cellular responses. The high affinity sigma receptor photoprobe iodoazidococaine (IAC) has labeled a 26-kDa protein in rat liver, rat brain, and human placenta (6Kahoun J.R. Ruoho A.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1393-1397Crossref PubMed Scopus (57) Google Scholar). Cloning studies have confirmed that both human and rodent sigma receptor cDNAs encode a 25.3-kDa protein (7Kekuda R. Prasad P.D. Fei Y.-J. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1996; 229: 553-558Crossref PubMed Scopus (393) Google Scholar, 8Hanner M. Moebius F.F. Flandorfer A. Knaus H.-G. Striessnig J. Kempner E. Glossman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8072-8077Crossref PubMed Scopus (832) Google Scholar, 9Seth P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1997; 241: 535-540Crossref PubMed Scopus (185) Google Scholar, 10Seth P. Fei Y.J. Li H.W. Huang W. Leibach F.H. Ganapathy V. J. Neurochem. 1998; 70: 922-931Crossref PubMed Scopus (280) Google Scholar). The protein encoded for by these cDNAs binds sigma receptor ligands, but physiological responses of expressed sigma receptors have yet to be demonstrated. The amino acid sequence deduced from these clones is not easily reconciled with a physiological function of ion channel modulation. The vast majority of receptors that couple to separate ion channel proteins contain seven putative membrane-spanning segments and require G-proteins for signal transduction (11Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Evidence both for (1Su T.-P. Crit. Rev. Neurobiol. 1993; 7: 187-203PubMed Google Scholar, 4Soriani O. Vaudry H. Mei Y.A. Roman F. Cazin L. J. Pharmacol. Exp. Ther. 1998; 286: 163-171PubMed Google Scholar) and against (12Morio Y. Tanimoto H. Yakushiji T. Morimoto Y. Brain Res. 1994; 637: 190-196Crossref PubMed Scopus (22) Google Scholar) a role for G-proteins in sigma receptor responses has been presented, but hydropathy analysis of the deduced sigma receptor sequence indicated that this protein contains a single putative membrane-spanning domain, with no regions known to interact with G-proteins. In fact, the proteins encoded for by sigma receptor cDNAs are novel. There are no vertebrate homologues, and the only known proteins with significant homology are fungal sterol isomerases. Furthermore, the sigma receptor contains an endoplasmic reticulum retention sequence. Thus, the structure of the sigma receptor raises the question of whether it is found on the plasma membrane and whether it is physically capable of modulating ion channels. A number of clonal cell lines contain sigma receptors (13Wu X.-Z. Bell J.A. Spivak C.E. London E.D. Su T.-P. J. Pharmacol. Exp. Ther. 1991; 257: 351-359PubMed Google Scholar, 14Kahoun J.R. Ruoho A.E. Kamenka J.-M. Domino E.F. Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, MI1992: 273-286Google Scholar, 15Ramamoorthy J.D. Ramamoorthy S. Mahesh V.B. Leibach F.H. Ganapathy V. Endocrinology. 1995; 136: 924-932Crossref PubMed Scopus (56) Google Scholar, 16Brent P.J. Pang G. Little G. Dosen P.J. VanHelden D.F. Biochem. Biophys. Res. Commun. 1996; 219: 219-226Crossref PubMed Scopus (60) Google Scholar). One of these, DMS-114, is derived from human small cell lung carcinoma and has been shown to release neurohypophysial peptides (17Pettingill O.S. Sorenson G.D. Wurster-Hill D.H. Curphy T.J. Noll W.W. Cate C.C. Maurer L.H. Cancer. 1980; 45: 906-918Crossref PubMed Scopus (153) Google Scholar, 18Sorenson G.D. Pettingill O.S. Binck-Johnson T. Cate C.C. Maurer L.H. Cancer. 1981; 47: 1289-1296Crossref PubMed Scopus (166) Google Scholar). Because our own work on sigma receptors began in the neurohypophysis (5Wilke R.A. Lupardus P.J. Grandy D.K. Rubinstein M. Low M.J. Jackson M.B. J. Physiol. (Lond.). 1999; 517: 391-406Crossref Scopus (58) Google Scholar), we became interested in using DMS-114 cells to investigate molecular aspects of sigma receptor function in greater detail. We found that DMS-114 cells are amenable to both photolabeling and patch clamp recording. This enabled us to test the hypothesis that the 26-kDa protein is involved in the modulation of I K by sigma receptor ligands. Further experiments showed that G-proteins do not mediate this response. Thus, this novel receptor protein employs a signal transduction mechanism not yet encountered in the ligand-induced modulation of ion channels. DMS-114 cells were obtained from the ATCC, Manassas, VA and maintained in Waymouth Medium 752/1 (ICN Biomedicals, Costa Mesa, CA) with 10% bovine calf serum (Life Technologies, Inc.). Flasks were incubated at 37 °C in 5% CO2, 95% air and subcultured regularly by mechanical dissociation. [125I]IAC was prepared according to Kahoun and Ruoho (6Kahoun J.R. Ruoho A.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1393-1397Crossref PubMed Scopus (57) Google Scholar). For photolabeling, DMS-114 cells were pelleted by low speed centrifugation (200 × g) and resuspended in phosphate-buffered saline (PBS) diluted 10-fold in distilled water. (PBS had the following composition: 140 mmNaCl, 2.7 mm KCl, 10 mmNa2HPO4, 1.8 mmKH2PO4, pH 7.3.) Recombinant DNase (1 IU) was then added, and cells were homogenized with a Teflon pestle. The homogenate was resuspended in PBS and divided into 100-μl aliquots. Sigma receptor ligands such as SKF10047 were then added, and the mixture was incubated for 30 min on ice. [125I]IAC (1 nm) was added, and the incubation was continued for an additional 7.5 min, at which point illumination was then performed for 5 s with a high pressure AH-mercury lamp. Proteins were separated by SDS-polyacrylamide gel electrophoresis (12% acrylamide) and scanned for [125I]IAC photolabeling on a PhosphorImager. Permanent autoradiograms were developed on x-ray film for all experiments included in this study. DMS-114 cells were plated on coverslips for voltage clamp recording. 2 h prior to recording, coverslips were transferred from the CO2/air incubator to a superfusion chamber containing physiological salt solution (115 mm NaCl, 4.0 mm KCl, 1.25 mmNaH2PO4, 26 mm NaHCO3, 2 mm CaCl2, 1 mm MgCl2, and 10 mm glucose, pH 7.4) saturated with 95% O2, 5% CO2. Individual cells were visualized with an upright differential interference contrast microscope (Diastar, Leica Microsystems, Inc., Buffalo, NY) and a × 40 water immersion, long working distance objective. Voltage clamp recordings were made with an EPC-9 patch clamp amplifier (InstruTECH Corp., Port Washington, NY) interfaced to a MacIntosh computer. Whole-cell currents were recorded using patch pipettes filled with 130 mm KCl, 10 mm EGTA, 2 mmMgCl2, 4 mm MgATP, 100 μm NaGTP, and 10 mm HEPES, pH 7.3. Pipettes were fabricated from thin walled borosilicate glass, and the pipette shanks were coated with Sylgard to reduce electrode capacitance (19Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15145) Google Scholar). Prior to approaching the cell membrane, pipette resistances typically ranged from 4 to 8 megaohms. Immediately after breaking in, cell capacitance and series resistance were determined with the transient cancellation capability of the EPC-9. In cases where the series resistance exceeded 15 megaohms, this value was partially compensated electronically. Sigma receptor ligands (other than IAC) were obtained from Research Biochemicals, International (Natick, MA). These compounds were dissolved in physiological buffer and added to the bathing solution by direct pipette injection or through the use of a simple gravity-feed system (rate, ∼2 ml/min). Prior to the addition of drugs, I K was recorded at 15-s intervals for 1–3 min to obtain a stable base line. I K was also recorded after the removal of any drug(s) to demonstrate viability of the cell and recovery of current to base line. In experiments conducted using highly lipophilic drugs such as (±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin (PPHT), the agent was first dissolved in Me2SO and then diluted into physiological saline to obtain the desired final drug concentration. The concentration of Me2SO never exceeded 0.1%; this vehicle, without drug(s), was tested previously and found to have no effect on I K (20Wilke R.A. Hsu S.-F. Jackson M.B. J. Pharmacol. Exp. Ther. 1998; 284: 542-548PubMed Google Scholar). Current recordings were analyzed on a MacIntosh computer with the computer program Pulse + PulseFit (InstruTECH Corp.). This program was used to fit current decays to exponential functions. The computer program Origin (Microcal, Northampton, MA) was used on a personal computer to fit concentration-response data to the following function:E = (100 − E max)/(1 +C/EC50) + E max, whereE denotes I K expressed as a percent of predrug control, C denotes the concentration of ligand in the superfusion medium, E max denotes response at saturating ligand, and EC50 denotes the concentration of drug producing a half-maximal effect (defined by the condition (E − 50)/E max = ½). Conductance-voltage plots were fitted to the following Boltzmann function: G=Gmin+(Gmax−Gmin)(1+e(V−V12)/k) (21Bielefeldt K. Rotter J.L. Jackson M.B. J. Physiol. (Lond.). 1992; 458: 41-67Crossref Scopus (66) Google Scholar). All four parameters, G min,G max, k, and V12 were varied to achieve the best fit. G min andG max represent conductance asymptotes at negative and positive prepulse potentials, respectively, kis the steepness factor, and V12 is the midpoint for voltage dependence. Simple statistical analyses were performed using the program Microsoft Excel. When arithmetic means were computed, they were presented with the S.E. All null hypotheses were subjected to a Student's t test, and a level of p < 0.05 was considered statistically significant. DMS-114 cells displayed an outward current in response to depolarizing test pulses under voltage clamp (Fig.1). Voltage steps from −80 to 10 mV rapidly activated this current, which showed a slight exponential decline as the potential was held constant for 500 ms. Tail currents reversed direction at the K+ equilibrium potential, and this value shifted appropriately with changes in external K+ concentration, indicating that the outward current was carried by K+ (n = 3, data not shown). When DMS-114 cells were exposed to the sigma receptor agonist SKF10047, whole-cell I K was inhibited in a concentration-dependent fashion (Fig. 1 A). Kinetic analysis revealed that the current amplitude was uniformly reduced by this agent and that the time constants for channel activation and inactivation remained unaltered (control: τact = 9.7 ± 0.6 ms, τinact = 3.8 ± 1.7 s; 10 μm SKF10047: τact = 9.7 ± 3.2 ms, τinact = 3.8 ± 1.0 s; n = 3). Whole-cell I K was also potently inhibited by the sigma receptor ligand IAC (Fig. 1 B). A radiolabeled version of this agent was originally developed as a photoaffinity label for neuronal sigma receptors (6Kahoun J.R. Ruoho A.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1393-1397Crossref PubMed Scopus (57) Google Scholar, 14Kahoun J.R. Ruoho A.E. Kamenka J.-M. Domino E.F. Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, MI1992: 273-286Google Scholar). Although significant inhibition ofI K was seen with 0.01 μm IAC, 1 μm IAC inhibited I K by more than 90%. The observation that IAC modulates I Kindicates that this compound is a potent sigma receptor agonist. To address the issue of ligand selectivity, additional chemically unrelated sigma receptor ligands were tested for inhibition ofI K. SKF10047 is the ligand for which sigma receptors were initially named (22Martin W.R. Eades C.G. Thompson J.A. Huppler R.E. Gilbert P.E. J. Pharmacol. Exp. Ther. 1976; 197: 517-532PubMed Google Scholar). Fig. 1 demonstrates that this compound inhibits I K in a concentration-dependent manner, and Fig.2 shows that this effect is reversible. Like SKF10047, DTG binds to sigma receptors with aK D in the nanomolar range, and its affinity for members of other receptor families is on the order of 1000-fold lower (1Su T.-P. Crit. Rev. Neurobiol. 1993; 7: 187-203PubMed Google Scholar). Fig. 2 demonstrates that DTG reduced I Kwith an efficacy and potency similar to SKF10047. Fig. 2 shows a similar result with PPHT, a drug that binds both sigma receptors and dopamine receptors (23Kebabian J.W. Tarazi F.I. Kula N.S. Baldessarini R.J. Drug Discovery Today. 1997; 2: 333-340Crossref Scopus (71) Google Scholar) and that has recently been shown to modulate neurohypophysial I K (5Wilke R.A. Lupardus P.J. Grandy D.K. Rubinstein M. Low M.J. Jackson M.B. J. Physiol. (Lond.). 1999; 517: 391-406Crossref Scopus (58) Google Scholar, 20Wilke R.A. Hsu S.-F. Jackson M.B. J. Pharmacol. Exp. Ther. 1998; 284: 542-548PubMed Google Scholar). Thus, four compounds known to bind sigma receptors, IAC, SKF10047, DTG, and PPHT, all inhibit I K in DMS-114 cells. [125I]IAC covalently labels sigma receptors upon illumination and was used to identify the receptor as a 26-kDa protein in rat brain and liver and in human placenta (6Kahoun J.R. Ruoho A.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1393-1397Crossref PubMed Scopus (57) Google Scholar). The labeling of this protein in each tissue could be blocked by haloperidol as well as other sigma receptor ligands. As noted above, IAC inhibited I K (Fig. 1). When DMS-114 cells were homogenized and illuminated in the presence of [125I]IAC, subsequent SDS-polyacrylamide gel electrophoresis and phosphorimaging demonstrated labeling of a protein with an apparent molecular mass of 26 kDa (Fig.3). Photolabeling of the 26-kDa band in DMS-114 cells was inhibited by two sigma receptor ligands, haloperidol (lane 2) and SKF10047 (lanes 4–6). This indicates that DMS-114 cells contain a protein with a similar molecular weight and that this protein has binding properties similar to previously characterized sigma receptors. A progressive increase in the block of [I125]IAC photolabeling of the 26-kDa band was evident as the SKF10047 concentration increased from 1 to 100 μm (Fig.4, top). The inhibitory effect of SKF10047 on the photolabeling of this band was plotted simultaneously with the inhibitory effect of SKF10047 onI K (Fig. 4, bottom). These effects had similar concentration dependences. When these data were fitted to a single-site saturation equation (see “Experimental Procedures”), an EC50 of 7 ± 3 μm was obtained for the inhibition of photolabeling, and an EC50 of 14 ± 3 μm was obtained for the inhibition ofI K. These two values are statistically indistinguishable, indicating that the inhibition ofI K is mediated by the 26-kDa sigma receptor identified by photolabeling. This is an important result because it links the 26-kDa sigma ligand binding protein to the functional response of I K modulation. Interestingly, the overall efficacy of SKF10047 appears to be identical for the two end points of inhibition of photolabeling and inhibition ofI K. At 100 μm, SKF10047 reduced whole-cell I K to 24 ± 5% of control (n = 3); this same concentration of SKF10047 reduced [125I]IAC photolabeling to 31 ± 8% of control (n = 3). This suggests that full occupation of sigma receptor binding sites produces a nearly complete block ofI K. Receptor-mediated modulation of voltage-gated ion channels often reflects a shift in the voltage dependence of the channel. Although this feature is not diagnostic of a particular transduction mechanism, it is still widely regarded as important. We therefore examined the voltage dependence of inhibition of I K by PPHT by varying test pulses used to activate I K from −60 to 30 mV. Current was recorded before and after a 3-min exposure to 30 μm PPHT. At all voltages tested, PPHT reduced I K by approximately proportional amounts, suggesting that sigma receptor ligands do not produce their inhibitory effect on K+channels by shifting the voltage dependence of activation. Current was converted to conductance using the relation, G K= I K/[V − εK], where G K is K+ conductance and εK is the Nernst potential for K+ computed for the bathing and patch pipette solution compositions. The plots ofG K versus pulse potential are shown in Fig. 5 along with best fitting Boltzmann functions (see “Experimental Procedures”). Under control conditions, the conductance-voltage plot was characterized by a steepness factor (k) of 10 ± 1 mV and a voltage midpoint (V12) of −6 ± 1 mV. After exposure to PPHT, neither the steepness factor (6 ± 4 mV) nor the voltage midpoint (−9 ± 4 mV) had been altered (n = 3). The modulation of I K in neurohypophysial nerve terminals by sigma receptor ligands exhibits a similar voltage independence (5Wilke R.A. Lupardus P.J. Grandy D.K. Rubinstein M. Low M.J. Jackson M.B. J. Physiol. (Lond.). 1999; 517: 391-406Crossref Scopus (58) Google Scholar, 20Wilke R.A. Hsu S.-F. Jackson M.B. J. Pharmacol. Exp. Ther. 1998; 284: 542-548PubMed Google Scholar). DMS-114 cell I K exhibits a weak voltage-dependent inactivation, as indicated by the decay of whole-cell current (Fig. 1), and it was already noted above that the time constants for activation and inactivation remained the same during challenges with sigma receptor ligand. We also examined inactivation ofI K by varying the voltage at which cells were held prior to depolarizing test pulses (to 10 mV). PPHT produced no shifts in the voltage dependence of inactivation (data not shown). These results indicate that sigma receptor-mediated modulation ofI K is not the result of shifts in voltage dependence. Extensive literature on receptor-mediated modulation of ion channels has shown that in most instances such responses are mediated by G-proteins (11Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 24Breitwieser G.E. J. Membr. Biol. 1996; 152: 1-11Crossref PubMed Scopus (40) Google Scholar, 25Schneider T. Igelmund P. Hescheler J. Trends Pharmacol. Sci. 1997; 18: 8-11Abstract Full Text PDF PubMed Scopus (52) Google Scholar). However, the deduced amino acid sequence of sigma receptors does not fit with a G-protein-coupled receptor motif (7Kekuda R. Prasad P.D. Fei Y.-J. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1996; 229: 553-558Crossref PubMed Scopus (393) Google Scholar, 8Hanner M. Moebius F.F. Flandorfer A. Knaus H.-G. Striessnig J. Kempner E. Glossman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8072-8077Crossref PubMed Scopus (832) Google Scholar, 9Seth P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1997; 241: 535-540Crossref PubMed Scopus (185) Google Scholar, 10Seth P. Fei Y.J. Li H.W. Huang W. Leibach F.H. Ganapathy V. J. Neurochem. 1998; 70: 922-931Crossref PubMed Scopus (280) Google Scholar). There have been reports that the binding activity of sigma receptors can be altered by GTP and guanosine nucleotides and that sigma receptor-mediated responses are attenuated by cholera toxin and GDPβS (1Su T.-P. Crit. Rev. Neurobiol. 1993; 7: 187-203PubMed Google Scholar, 4Soriani O. Vaudry H. Mei Y.A. Roman F. Cazin L. J. Pharmacol. Exp. Ther. 1998; 286: 163-171PubMed Google Scholar), but another study showed that cholera toxin had no effect (12Morio Y. Tanimoto H. Yakushiji T. Morimoto Y. Brain Res. 1994; 637: 190-196Crossref PubMed Scopus (22) Google Scholar). Both the size (1Su T.-P. Crit. Rev. Neurobiol. 1993; 7: 187-203PubMed Google Scholar) and deduced amino acid sequence (7Kekuda R. Prasad P.D. Fei Y.-J. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1996; 229: 553-558Crossref PubMed Scopus (393) Google Scholar, 8Hanner M. Moebius F.F. Flandorfer A. Knaus H.-G. Striessnig J. Kempner E. Glossman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8072-8077Crossref PubMed Scopus (832) Google Scholar, 9Seth P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1997; 241: 535-540Crossref PubMed Scopus (185) Google Scholar, 10Seth P. Fei Y.J. Li H.W. Huang W. Leibach F.H. Ganapathy V. J. Neurochem. 1998; 70: 922-931Crossref PubMed Scopus (280) Google Scholar) of sigma receptors are difficult to reconcile with that of known G-protein-coupled receptors. We therefore tested the role of G-proteins by adding GDPβS (100 μm) to the patch pipette filling solution and allowing it to diffuse into the cell interior during whole-cell recordings. Base-line currents were first collected for at least 3 min after break in, allowing ample time for a molecule of this size to perfuse the cell interior (26Pusch M. Neher E. Pfluegers Arch. Eur. J. Physiol. 1988; 411: 204-211Crossref PubMed Scopus (568) Google Scholar). G-protein-mediated responses in neurons have been shown to be attenuated 75% by this mode of GDPβS addition 2 min after break in (27Trussell L.O. Jackson M.B. J. Neurosci. 1987; 7: 3306-3316Crossref PubMed Google Scholar). We found that responses to SKF10047 were not reduced by GDPβS. Both 10 and 100 μm SKF10047 showed equal efficacy for the inhibition of I K regardless of whether cells were perfused with 100 μm GDPβS or 100 μm GTP (Fig. 6). It therefore appears that sigma receptor-mediated modulation ofI K can occur independently of G-protein activation. Similar results have been obtained with sigma receptor-mediated modulation of I K in rat neurohypophysial nerve terminals. 2P. J. Lupardus, R. A. Wilke, Y. Chen, R. E. Ruoho, and M. B. Jackson, submitted for publication. In these experiments GDPβS also failed to block the modulation ofI K by SKF10047. The entry of the guanine nucleotide GTPγS into neurohypophysial terminals was verified by monitoring current through Ca2+-activated K+channels. GTPγS triggers the G-protein-mediated dephosphorylation of this channel in the neurohypophysis (28Bielefeldt K. Jackson M.B. J. Physiol. (Lond.). 1994; 475: 241-254Crossref Scopus (88) Google Scholar), and 50 μmGTPγS added to the patch pipette filling solution reduced current to 79 ± 3% of the original level at break in, with a half-time of 29 ± 4 s (n = 11). These studies demonstrate that sigma receptor activation reduces voltage-dependent I K by binding to a protein with a molecular mass of 26 kDa. Four chemically distinct sigma receptor ligands (SKF10047, DTG, PPHT, and IAC) inhibitedI K in a reversible, concentration-dependent fashion. IAC also photolabeled a protein with a molecular mass of 26 kDa, and this photolabeling was blocked by concentrations of SKF10047 similar to those that inhibitedI K in patch clamp recordings. These studies provide the first link of a protein with sigma ligand binding activity to K+ channel modulation. Thus, despite the presence of a putative endoplasmic reticulum sequence and the absence of putativeN-glycosylation sites (7Kekuda R. Prasad P.D. Fei Y.-J. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1996; 229: 553-558Crossref PubMed Scopus (393) Google Scholar, 8Hanner M. Moebius F.F. Flandorfer A. Knaus H.-G. Striessnig J. Kempner E. Glossman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8072-8077Crossref PubMed Scopus (832) Google Scholar, 9Seth P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1997; 241: 535-540Crossref PubMed Scopus (185) Google Scholar, 10Seth P. Fei Y.J. Li H.W. Huang W. Leibach F.H. Ganapathy V. J. Neurochem. 1998; 70: 922-931Crossref PubMed Scopus (280) Google Scholar), the physiological function of the sigma receptor indicates a location in or near the plasma membrane. The modulation of voltage-dependent K+ channels by membrane-bound receptors has been extensively studied in many systems. In general, transduction of these responses requires the activation of a G-protein (11Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 24Breitwieser G.E. J. Membr. Biol. 1996; 152: 1-11Crossref PubMed Scopus (40) Google Scholar, 25Schneider T. Igelmund P. Hescheler J. Trends Pharmacol. Sci. 1997; 18: 8-11Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Our observation that the inhibition of I K is mediated by a receptor with an apparent molecular mass of only 26 kDa is difficult to reconcile with the hypothesis that G-proteins are involved in the transduction of this response. Many G-protein-coupled receptors are similar in size to rhodopsin, with molecular weights of roughly 40–50 kDa (plus carbohydrate) (29Ross E.M. Neuron. 1989; 3: 141-152Abstract Full Text PDF PubMed Scopus (258) Google Scholar). Although G-protein-coupled receptors with higher molecular weights are quite common (30Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar), homologous receptors with molecular masses as low as 26 kDa have yet to be identified. Furthermore, the seven transmembrane segments found in all known G-protein-coupled receptors are inconsistent with the single membrane-spanning segment implied by the hydropathy plots constructed from the deduced sigma receptor amino acid sequence (7Kekuda R. Prasad P.D. Fei Y.-J. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1996; 229: 553-558Crossref PubMed Scopus (393) Google Scholar, 8Hanner M. Moebius F.F. Flandorfer A. Knaus H.-G. Striessnig J. Kempner E. Glossman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8072-8077Crossref PubMed Scopus (832) Google Scholar, 9Seth P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1997; 241: 535-540Crossref PubMed Scopus (185) Google Scholar, 10Seth P. Fei Y.J. Li H.W. Huang W. Leibach F.H. Ganapathy V. J. Neurochem. 1998; 70: 922-931Crossref PubMed Scopus (280) Google Scholar). Efforts to establish a role for G-proteins in sigma receptor function have met with mixed results. Cholera toxin reduced responses to sigma receptor ligands in one study (4Soriani O. Vaudry H. Mei Y.A. Roman F. Cazin L. J. Pharmacol. Exp. Ther. 1998; 286: 163-171PubMed Google Scholar) but not in another (12Morio Y. Tanimoto H. Yakushiji T. Morimoto Y. Brain Res. 1994; 637: 190-196Crossref PubMed Scopus (22) Google Scholar). In melanotrophs, the guanine nucleotide analogue GDPβS has been shown to prevent the inhibition of I K by DTG (4Soriani O. Vaudry H. Mei Y.A. Roman F. Cazin L. J. Pharmacol. Exp. Ther. 1998; 286: 163-171PubMed Google Scholar). These results may reflect the existence of another molecular species of sigma receptor that is a member of the G-protein-coupled receptor family. In the present study, cells perfused internally with GDPβS exhibited the same response to SKF10047 as control cells perfused internally with GTP, and GDPβS is known to be a very potent inhibitor of G-protein-dependent processes (11Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 24Breitwieser G.E. J. Membr. Biol. 1996; 152: 1-11Crossref PubMed Scopus (40) Google Scholar, 25Schneider T. Igelmund P. Hescheler J. Trends Pharmacol. Sci. 1997; 18: 8-11Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 27Trussell L.O. Jackson M.B. J. Neurosci. 1987; 7: 3306-3316Crossref PubMed Google Scholar, 29Ross E.M. Neuron. 1989; 3: 141-152Abstract Full Text PDF PubMed Scopus (258) Google Scholar). This result therefore indicates that the recently cloned 26-kDa receptor modulates K+ channels without coupling to G-proteins. In this regard it is relevant that the concentration dependences of both ligand binding and I K inhibition by SKF10047 were similar (Fig. 4). In contrast to G-protein-coupled receptors, where spare receptors and other variations in the efficiency of coupling can cause large discrepancies between the apparentK D and the physiological EC50 (29Ross E.M. Neuron. 1989; 3: 141-152Abstract Full Text PDF PubMed Scopus (258) Google Scholar), the modulation of K+ channel function by sigma receptor ligands appears to be tightly coupled to receptor binding. If G-proteins do not mediate the inhibition ofI K by sigma receptor activation, then these results may indicate that sigma receptor-mediated signal transduction depends on other molecular factors. An interesting possibility is that sigma receptors alter K+ channel activity through a direct protein-protein interaction with the channel, analogous to the actions of auxiliary β subunits (31Adelman J.P. Curr. Opin. Neurobiol. 1995; 5: 286-295Crossref PubMed Scopus (34) Google Scholar) and MinK proteins (32Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1518) Google Scholar, 33Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romey G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1394) Google Scholar), both of which can modulate the function of voltage-gated K+channels without forming channels themselves. Another possibility is that sigma receptors interact with a protein kinase (by a G-protein-independent mechanism) and that this enzyme modifies channel function. These mechanisms have not been demonstrated previously in ligand-induced ion channel modulation, indicating that sigma receptors inhibit I K by a new transduction process. It is noteworthy that DMS-114 cells are tumor cells derived from a neuroendocrine progenitor (34Devita V.T. Hellman S. Rosenberg S.A. Cancer: Principles and Practice of Oncology. 5th Ed. Lippincott-Raven, Philadelphia1997Google Scholar). These cells retain many properties of an excitable cell line, including the expression of voltage-dependent ion channels (35Pancrazio J.J. Tabbara I.A. Kim Y.I. Anticancer Res. 1993; 13: 1231-1234PubMed Google Scholar, 36Morton M.E. Cassidy T.N. Froehner S.C. Gilmour S.C. Laurens R.L. FASEB J. 1994; 8: 884-888Crossref PubMed Scopus (7) Google Scholar). The coupling between sigma receptors and I K described here in DMS-114 cells is very similar to that found in neuroendocrine neurohypophysial nerve terminals (5Wilke R.A. Lupardus P.J. Grandy D.K. Rubinstein M. Low M.J. Jackson M.B. J. Physiol. (Lond.). 1999; 517: 391-406Crossref Scopus (58) Google Scholar). Furthermore, DMS-114 cells exhibit ectopic release of the neurohypophysial hormone vasopressin (17Pettingill O.S. Sorenson G.D. Wurster-Hill D.H. Curphy T.J. Noll W.W. Cate C.C. Maurer L.H. Cancer. 1980; 45: 906-918Crossref PubMed Scopus (153) Google Scholar, 18Sorenson G.D. Pettingill O.S. Binck-Johnson T. Cate C.C. Maurer L.H. Cancer. 1981; 47: 1289-1296Crossref PubMed Scopus (166) Google Scholar). The coexpression of these distinctive properties in both DMS-114 cells and the neurohypophysis may reflect a link in the mechanisms of regulation of different cellular functions that is operating both in this tumor cell line and in the native hypothalamic neurohypophysial system. In vivo, tumor-related secretion of ectopic neurohypophysial hormones is responsible for inducing derangements of fluid and sodium homeostasis in as many as 30% of patients with primary small cell lung carcinoma (37Johnson B.E. Chute J.P. Rushin J. Williams J. Tram Le P. Venzon D. Richardson G.E. Am. J. Respir. Crit. Care Med. 1997; 156: 1669-1678Crossref PubMed Scopus (74) Google Scholar). The finding that sigma receptors are functionally linked to membrane excitability in such cells indicates that this protein may represent a useful therapeutic target for the curtailment of ectopic hormone secretion. We thank Dr. Bradford Schwartz for providing the DMS-114 cells and Drs. Gerard Ahern and Sujata Swaminathan for help in the design of the experiments." @default.
• W2056581302 created "2016-06-24" @default.
• W2056581302 creator A5046231847 @default.
• W2056581302 creator A5046833465 @default.
• W2056581302 creator A5050603140 @default.
• W2056581302 creator A5051487266 @default.
• W2056581302 creator A5068754205 @default.
• W2056581302 creator A5075764403 @default.
• W2056581302 date "1999-06-01" @default.
• W2056581302 modified "2023-10-03" @default.
• W2056581302 title "Sigma Receptor Photolabeling and Sigma Receptor-mediated Modulation of Potassium Channels in Tumor Cells" @default.
• W2056581302 cites W1496211185 @default.
• W2056581302 cites W1594866681 @default.
• W2056581302 cites W1981337088 @default.
• W2056581302 cites W1983137097 @default.
• W2056581302 cites W1987749939 @default.
• W2056581302 cites W1994224293 @default.
• W2056581302 cites W2001614713 @default.
• W2056581302 cites W2002422701 @default.
• W2056581302 cites W2002957477 @default.
• W2056581302 cites W2006283790 @default.
• W2056581302 cites W2009667219 @default.
• W2056581302 cites W2015063786 @default.
• W2056581302 cites W2018688747 @default.
• W2056581302 cites W2020425432 @default.
• W2056581302 cites W2026692772 @default.
• W2056581302 cites W2033388874 @default.
• W2056581302 cites W2036991975 @default.
• W2056581302 cites W2040766257 @default.
• W2056581302 cites W2050400539 @default.
• W2056581302 cites W2054597500 @default.
• W2056581302 cites W2055876432 @default.
• W2056581302 cites W2063441238 @default.
• W2056581302 cites W2064514265 @default.
• W2056581302 cites W2071665493 @default.
• W2056581302 cites W2108618509 @default.
• W2056581302 cites W2147646436 @default.
• W2056581302 cites W2162213293 @default.
• W2056581302 doi "https://doi.org/10.1074/jbc.274.26.18387" @default.
• W2056581302 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10373444" @default.
• W2056581302 hasPublicationYear "1999" @default.
• W2056581302 type Work @default.
• W2056581302 sameAs 2056581302 @default.
• W2056581302 citedByCount "66" @default.
• W2056581302 countsByYear W20565813022012 @default.
• W2056581302 countsByYear W20565813022013 @default.
• W2056581302 countsByYear W20565813022014 @default.
• W2056581302 countsByYear W20565813022015 @default.
• W2056581302 countsByYear W20565813022016 @default.
• W2056581302 countsByYear W20565813022017 @default.
• W2056581302 countsByYear W20565813022018 @default.
• W2056581302 countsByYear W20565813022019 @default.
• W2056581302 countsByYear W20565813022020 @default.
• W2056581302 countsByYear W20565813022021 @default.
• W2056581302 countsByYear W20565813022023 @default.
• W2056581302 crossrefType "journal-article" @default.
• W2056581302 hasAuthorship W2056581302A5046231847 @default.
• W2056581302 hasAuthorship W2056581302A5046833465 @default.
• W2056581302 hasAuthorship W2056581302A5050603140 @default.
• W2056581302 hasAuthorship W2056581302A5051487266 @default.
• W2056581302 hasAuthorship W2056581302A5068754205 @default.
• W2056581302 hasAuthorship W2056581302A5075764403 @default.
• W2056581302 hasBestOaLocation W20565813021 @default.
• W2056581302 hasConcept C103133801 @default.
• W2056581302 hasConcept C121332964 @default.
• W2056581302 hasConcept C123079801 @default.
• W2056581302 hasConcept C12554922 @default.
• W2056581302 hasConcept C170493617 @default.
• W2056581302 hasConcept C178790620 @default.
• W2056581302 hasConcept C185592680 @default.
• W2056581302 hasConcept C24890656 @default.
• W2056581302 hasConcept C2777052854 @default.
• W2056581302 hasConcept C2778049214 @default.
• W2056581302 hasConcept C2778938600 @default.
• W2056581302 hasConcept C517785266 @default.
• W2056581302 hasConcept C55493867 @default.
• W2056581302 hasConcept C62520636 @default.
• W2056581302 hasConcept C83743174 @default.
• W2056581302 hasConcept C86803240 @default.
• W2056581302 hasConcept C95444343 @default.
• W2056581302 hasConceptScore W2056581302C103133801 @default.
• W2056581302 hasConceptScore W2056581302C121332964 @default.
• W2056581302 hasConceptScore W2056581302C123079801 @default.
• W2056581302 hasConceptScore W2056581302C12554922 @default.
• W2056581302 hasConceptScore W2056581302C170493617 @default.
• W2056581302 hasConceptScore W2056581302C178790620 @default.
• W2056581302 hasConceptScore W2056581302C185592680 @default.
• W2056581302 hasConceptScore W2056581302C24890656 @default.
• W2056581302 hasConceptScore W2056581302C2777052854 @default.
• W2056581302 hasConceptScore W2056581302C2778049214 @default.
• W2056581302 hasConceptScore W2056581302C2778938600 @default.
• W2056581302 hasConceptScore W2056581302C517785266 @default.
• W2056581302 hasConceptScore W2056581302C55493867 @default.
• W2056581302 hasConceptScore W2056581302C62520636 @default.
• W2056581302 hasConceptScore W2056581302C83743174 @default.
• W2056581302 hasConceptScore W2056581302C86803240 @default.
• W2056581302 hasConceptScore W2056581302C95444343 @default.
• W2056581302 hasIssue "26" @default.

• next