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MTSEA potentiates 5-HT3 receptors containing the nicotinic α4 subunit
Neuropharmacology 38 (1999) 1913 – 1915 www.elsevier.com/locate/neuropharm
Rapid communication
MTSEA potentiates 5-HT3 receptors containing the nicotinic a4 subunit Steve Kriegler, Sterling Sudweeks, Jerrel L. Yakel * Laboratory of Signal Transduction, National Institute of En6ironmental Health Sciences, National Institutes of Health, P.O. Box 12233, 111 T.W. Alexander Dri6e, Research Triangle Park, N.C. 27709, USA Accepted 4 June 1999
Abstract In order to study the subunit composition of 5-HT3 receptors (5-HT3R), we report that (2-aminoethyl)methanethiosulfonate (MTSEA) can enhance the function of both nicotinic ACh receptors (nAChRs) comprised of a4/b2 subunits, and heteromeric channels assembled from serotonin 5-HT3R and a4 nAChR subunits. MTSEA has no effect on homomeric 5-HT3 receptor channels. Published by Elsevier Science Ltd.
1. Introduction Using a variety of techniques, we have shown that the serotonin 5-HT3 receptor (5-HT3R) subunit can co-assemble with the a4 nicotinic ACh receptor (nAChR) subunit in heterologous expression systems (van Hooft et al., 1998). Furthermore, we have recently demonstrated that the a4 nAChR subunit forms part of the ion channel pore along with the 5-HT3R subunit (Kriegler et al., 1999). With the long-range aim to explore whether the co-assembly of the 5-HT3R and a4 nAChR subunits occurs in vivo, we examined one compound for its potential to detect the co-assembly of wildtype (i.e. non-mutated) subunits into functional receptors. MTSEA ((2-aminoethyl)methanethiosulfonate), a positively charged sulfhydryl reagent that reacts with cysteine residues, has previously been reported to potentiate the current and alter the rate of desensitization of wildtype a4/b2 nAChRs (Yu et al., 1996). Therefore we tested whether MTSEA enhances heteromeric receptor function composed of the a4 nAChR and 5-HT3R subunits in Xenopus oocytes.
* Corresponding author. Tel.: +1-919-5411407; fax: + 1-9195411898. E-mail address: [email protected] (J.L. Yakel) 0028-3908/99/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 0 9 - 4
Xenopus lae6is oocytes were prepared and maintained as described previously (Kriegler et al., 1999). Oocytes were defoliculated with collagenase B and injected with RNA. The total amount of RNA injected for each subunit was (in ng) 5-HT3R (0.5), a4 and b2 nAChR subunits (25). Experiments were performed 1 to 4 days after injection. The 5-HT3R plasmid (long form) was kindly provided by D. Julius, and the rat nAChR a4 and b2 plasmids were kindly provided by J. Patrick. Current responses were obtained by two-electrode voltage-clamp recording at a holding potential of −25 or −60 mV, using a Geneclamp 500 amplifier (Axon Instruments). Electrodes contained 3 M KCl with 0.4 M BAPTA and had resistances of B 0.5 MV. ACh (Sigma) and 5-HT (RBI) were freshly prepared in bath solution from a frozen stock, and applied via a synthetic quartz perfusion tube (0.7 mm) operated by a computer controlled valve. Recordings were performed in a solution containing (in mM) NaCl (96), KCl (2), CaCl2 (1.8), MgCl2 (1), HEPES (10) and niflumic acid (0.3). Oocytes were maintained in culture in the same solution, without niflumic acid, and with the addition of 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 mg/ml gentamycin and 5% horse serum. MTSEA (Toronto Research Chemicals) was dissolved in solution immediately prior to application and used in less than 2 min.
1914
S. Kriegler et al. / Neuropharmacology 38 (1999) 1913–1915
In preliminary experiments, we studied the effect of MTSEA on wildtype a4/b2 nAChR channels. After establishing a stable baseline current, MTSEA (2.5 or 5 mM) was added for 5 min. We then measured the difference in amplitudes of the acetylcholine (ACh)-activated current (5 mM ACh) after washing the oocyte for 2 min. This treatment resulted in a large, but relatively slow, increase in the response amplitude (6139 88%, n=5; mean9standard error); this value was reached after approx. 15 min. These results are consistent with those of Yu et al. (1996) where MTSEA was found to increase the open channel time. Next we tested whether MTSEA (15 mM for 10 min) had any effect on oocytes expressing the 5-HT3R subunit. The control recordings shown in Fig. 1A and C demonstrate that MTSEA has no significant effect on either the amplitude or the rate of desensitization of homomeric 5-HT3R channels. However when the 5HT3R subunit was co-expressed with the wildtype a4 nAChR subunit, MTSEA significantly slowed the rate of desensitization (P B 0.04). In 11 cells, the mean time
required for the response to decay by 50% (t1) in the 2 continued presence of agonist was 0.7390.1 s, the value of t1 increased by 124 949% after MTSEA treat2 ment. The mean amplitude of the 5-HT3R response also appeared to increase, however this change was not statistically significant (P= 0.09 calculated using a twotailed t-test with unequal variance.) Since the rate of desensitization of the 5-HT3R is dependent on membrane potential (Jackson and Yakel, 1995), we repeated these experiments at a holding potential of − 25 mV rather than −60 mV. At this potential, the current did not decay to 50% of its peak amplitude during the 4 s application of 5-HT. Under these conditions, MTSEA had no effect on either the peak amplitude (29 2%; n=3) or the area under the curve (29 3%). These results demonstrate that MTSEA can distinguish whether the a4 nAChR subunit co-assembles with the 5-HT3R subunit. In a variety of native tissues, 5-HT3Rs exhibit a diverse array of pharmacological and physiological properties suggesting that these receptors are comprised of a heterologous array of 5HT3R subunits (Fletcher and Barnes 1998; Davies et al., 1999). Perhaps co-assembly between the 5-HT3R and a4 nAChR subunits might explain some of the diverse functional properties of native 5-HT3Rs, however it should be noted that Fletcher et al. (1998) reported that 5-HT3Rs purified from porcine brain did not contain the a4 nAChR subunit. Nevertheless in light of the demonstrated interaction between the serotonin 5-HT3R and a4 nAChR subunits in heterologous expression systems, we propose that MTSEA may serve as a valuable pharmacological tool that might help to determine whether the co-assembly between these two subunits from different neurotransmitter receptors types may actually occur in vivo.
Acknowledgements We would like to thank D. Armstrong and D. Pettit for advice in preparing the manuscript and D. Julius and J. Patrick for providing us with plasmid DNA.
References
Fig. 1. MTSEA enhances the current of 5-HT3Rs assembled from wildtype subunits. Traces are from oocytes expressing 5-HT3R subunits, injected alone (A) or together with (B) the wildtype a4 nAChR subunit in response to 50 mM serotonin (indicated by the horizontal bar). MTSEA (15 mM) was applied for 10 min, and the second trace was recorded 15 min after the wash out of MTSEA. The holding potential was − 60 mV, and the average change is shown in C (mean9 S.E.)
Davies, P.A., Pistis, M., Hanna, M.C., Peters, J.A., Lambert, J.J., Hales, T.G., Kirkness, E.F., 1999. The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature 377, 359–363. Fletcher, S., Barnes, N.M., 1998. Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes? Trends Pharmacol. Sci. 19, 212 – 215. Fletcher, S., Lindstrom, J.M., McKernan, R.M., Barnes, N.M., 1998. Evidence that porcine native 5-HT3 receptors do not contain nicotinic acetylcholine receptor subunits. Neuropharmacology 37, 397 – 399.
S. Kriegler et al. / Neuropharmacology 38 (1999) 1913–1915 Jackson, M.B., Yakel, J.L., 1995. The 5-HT3 receptor channel. Ann. Rev. Physiol. 57, 447–468. Kriegler, S., Sudweeks, S., Yakel, J.L., 1999. The nicotinic a4 receptor subunits contributes to the lining of the ion channel pore when expressed with the 5-HT3 receptor subunit. J. Biol. Chem. 274, 3934 – 3936. van Hooft, J.A., Spier, A.D., Yakel, J.L., Lummis, S.C.R.,Vijver-
.
1915
berg, H.P.M., 1998. Promiscuous coassebly of serotonin 5HT3 and nictotinic a4 receptor subunits into Ca2 + permeable ion channels. Proc. Natl. Acad. Sci. USA 95, 11461– 11465. Yu, C., Ramirez-Latorre, J.A., Role, L., 1996. Ca2 + modulation of a4b2 and a4a5b2 combinations of neuronal nicotinic receptors. Soc. Neurosci. Abstr.. 603.10 22, 1526.
Rapid communication
MTSEA potentiates 5-HT3 receptors containing the nicotinic a4 subunit Steve Kriegler, Sterling Sudweeks, Jerrel L. Yakel * Laboratory of Signal Transduction, National Institute of En6ironmental Health Sciences, National Institutes of Health, P.O. Box 12233, 111 T.W. Alexander Dri6e, Research Triangle Park, N.C. 27709, USA Accepted 4 June 1999
Abstract In order to study the subunit composition of 5-HT3 receptors (5-HT3R), we report that (2-aminoethyl)methanethiosulfonate (MTSEA) can enhance the function of both nicotinic ACh receptors (nAChRs) comprised of a4/b2 subunits, and heteromeric channels assembled from serotonin 5-HT3R and a4 nAChR subunits. MTSEA has no effect on homomeric 5-HT3 receptor channels. Published by Elsevier Science Ltd.
1. Introduction Using a variety of techniques, we have shown that the serotonin 5-HT3 receptor (5-HT3R) subunit can co-assemble with the a4 nicotinic ACh receptor (nAChR) subunit in heterologous expression systems (van Hooft et al., 1998). Furthermore, we have recently demonstrated that the a4 nAChR subunit forms part of the ion channel pore along with the 5-HT3R subunit (Kriegler et al., 1999). With the long-range aim to explore whether the co-assembly of the 5-HT3R and a4 nAChR subunits occurs in vivo, we examined one compound for its potential to detect the co-assembly of wildtype (i.e. non-mutated) subunits into functional receptors. MTSEA ((2-aminoethyl)methanethiosulfonate), a positively charged sulfhydryl reagent that reacts with cysteine residues, has previously been reported to potentiate the current and alter the rate of desensitization of wildtype a4/b2 nAChRs (Yu et al., 1996). Therefore we tested whether MTSEA enhances heteromeric receptor function composed of the a4 nAChR and 5-HT3R subunits in Xenopus oocytes.
* Corresponding author. Tel.: +1-919-5411407; fax: + 1-9195411898. E-mail address: [email protected] (J.L. Yakel) 0028-3908/99/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 0 9 - 4
Xenopus lae6is oocytes were prepared and maintained as described previously (Kriegler et al., 1999). Oocytes were defoliculated with collagenase B and injected with RNA. The total amount of RNA injected for each subunit was (in ng) 5-HT3R (0.5), a4 and b2 nAChR subunits (25). Experiments were performed 1 to 4 days after injection. The 5-HT3R plasmid (long form) was kindly provided by D. Julius, and the rat nAChR a4 and b2 plasmids were kindly provided by J. Patrick. Current responses were obtained by two-electrode voltage-clamp recording at a holding potential of −25 or −60 mV, using a Geneclamp 500 amplifier (Axon Instruments). Electrodes contained 3 M KCl with 0.4 M BAPTA and had resistances of B 0.5 MV. ACh (Sigma) and 5-HT (RBI) were freshly prepared in bath solution from a frozen stock, and applied via a synthetic quartz perfusion tube (0.7 mm) operated by a computer controlled valve. Recordings were performed in a solution containing (in mM) NaCl (96), KCl (2), CaCl2 (1.8), MgCl2 (1), HEPES (10) and niflumic acid (0.3). Oocytes were maintained in culture in the same solution, without niflumic acid, and with the addition of 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 mg/ml gentamycin and 5% horse serum. MTSEA (Toronto Research Chemicals) was dissolved in solution immediately prior to application and used in less than 2 min.
1914
S. Kriegler et al. / Neuropharmacology 38 (1999) 1913–1915
In preliminary experiments, we studied the effect of MTSEA on wildtype a4/b2 nAChR channels. After establishing a stable baseline current, MTSEA (2.5 or 5 mM) was added for 5 min. We then measured the difference in amplitudes of the acetylcholine (ACh)-activated current (5 mM ACh) after washing the oocyte for 2 min. This treatment resulted in a large, but relatively slow, increase in the response amplitude (6139 88%, n=5; mean9standard error); this value was reached after approx. 15 min. These results are consistent with those of Yu et al. (1996) where MTSEA was found to increase the open channel time. Next we tested whether MTSEA (15 mM for 10 min) had any effect on oocytes expressing the 5-HT3R subunit. The control recordings shown in Fig. 1A and C demonstrate that MTSEA has no significant effect on either the amplitude or the rate of desensitization of homomeric 5-HT3R channels. However when the 5HT3R subunit was co-expressed with the wildtype a4 nAChR subunit, MTSEA significantly slowed the rate of desensitization (P B 0.04). In 11 cells, the mean time
required for the response to decay by 50% (t1) in the 2 continued presence of agonist was 0.7390.1 s, the value of t1 increased by 124 949% after MTSEA treat2 ment. The mean amplitude of the 5-HT3R response also appeared to increase, however this change was not statistically significant (P= 0.09 calculated using a twotailed t-test with unequal variance.) Since the rate of desensitization of the 5-HT3R is dependent on membrane potential (Jackson and Yakel, 1995), we repeated these experiments at a holding potential of − 25 mV rather than −60 mV. At this potential, the current did not decay to 50% of its peak amplitude during the 4 s application of 5-HT. Under these conditions, MTSEA had no effect on either the peak amplitude (29 2%; n=3) or the area under the curve (29 3%). These results demonstrate that MTSEA can distinguish whether the a4 nAChR subunit co-assembles with the 5-HT3R subunit. In a variety of native tissues, 5-HT3Rs exhibit a diverse array of pharmacological and physiological properties suggesting that these receptors are comprised of a heterologous array of 5HT3R subunits (Fletcher and Barnes 1998; Davies et al., 1999). Perhaps co-assembly between the 5-HT3R and a4 nAChR subunits might explain some of the diverse functional properties of native 5-HT3Rs, however it should be noted that Fletcher et al. (1998) reported that 5-HT3Rs purified from porcine brain did not contain the a4 nAChR subunit. Nevertheless in light of the demonstrated interaction between the serotonin 5-HT3R and a4 nAChR subunits in heterologous expression systems, we propose that MTSEA may serve as a valuable pharmacological tool that might help to determine whether the co-assembly between these two subunits from different neurotransmitter receptors types may actually occur in vivo.
Acknowledgements We would like to thank D. Armstrong and D. Pettit for advice in preparing the manuscript and D. Julius and J. Patrick for providing us with plasmid DNA.
References
Fig. 1. MTSEA enhances the current of 5-HT3Rs assembled from wildtype subunits. Traces are from oocytes expressing 5-HT3R subunits, injected alone (A) or together with (B) the wildtype a4 nAChR subunit in response to 50 mM serotonin (indicated by the horizontal bar). MTSEA (15 mM) was applied for 10 min, and the second trace was recorded 15 min after the wash out of MTSEA. The holding potential was − 60 mV, and the average change is shown in C (mean9 S.E.)
Davies, P.A., Pistis, M., Hanna, M.C., Peters, J.A., Lambert, J.J., Hales, T.G., Kirkness, E.F., 1999. The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature 377, 359–363. Fletcher, S., Barnes, N.M., 1998. Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes? Trends Pharmacol. Sci. 19, 212 – 215. Fletcher, S., Lindstrom, J.M., McKernan, R.M., Barnes, N.M., 1998. Evidence that porcine native 5-HT3 receptors do not contain nicotinic acetylcholine receptor subunits. Neuropharmacology 37, 397 – 399.
S. Kriegler et al. / Neuropharmacology 38 (1999) 1913–1915 Jackson, M.B., Yakel, J.L., 1995. The 5-HT3 receptor channel. Ann. Rev. Physiol. 57, 447–468. Kriegler, S., Sudweeks, S., Yakel, J.L., 1999. The nicotinic a4 receptor subunits contributes to the lining of the ion channel pore when expressed with the 5-HT3 receptor subunit. J. Biol. Chem. 274, 3934 – 3936. van Hooft, J.A., Spier, A.D., Yakel, J.L., Lummis, S.C.R.,Vijver-
.
1915
berg, H.P.M., 1998. Promiscuous coassebly of serotonin 5HT3 and nictotinic a4 receptor subunits into Ca2 + permeable ion channels. Proc. Natl. Acad. Sci. USA 95, 11461– 11465. Yu, C., Ramirez-Latorre, J.A., Role, L., 1996. Ca2 + modulation of a4b2 and a4a5b2 combinations of neuronal nicotinic receptors. Soc. Neurosci. Abstr.. 603.10 22, 1526.