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Low molecular weight mRNA encodes a protein that controls serotonin 5-HT1c and acetylcholine M1 receptor sensitivity
A E Walter1, J H Hoger, C Labarca
1Division of Biology, California Institute of Technology, Pasadena 91125.
Abstract:
Serotonin 5-HT1c and acetylcholine M1 receptors activate phosphoinositidase, resulting in an increased formation of IP3 and 1,2 diacylglycerol. In Xenopus oocytes injected with mRNA encoding either of these receptors, Ca2+ released from intracellular stores in response to IP3 then opens Ca(2+)-gated Cl-channels. In the present experiments, oocytes expressing a transcript from a cloned mouse serotonin 5-HT1c receptor were exposed to identical 15-s pulses of agonist, administered 2 min apart; the second current response was two to three times that of the first. However, in those oocytes coinjected with the 5-HT1c receptor transcript and a low molecular weight fraction (0.3-1.5 kb) of rat brain mRNA, the second current response was approximately 50% of the first. Thus, the low molecular weight RNA encodes a protein (or proteins) that causes desensitization. Experiments using fura-2 or a Ca(2+)-free superfusate indicated that desensitization of the 5-HT1c receptor response does not result from a sustained elevation of intracellular Ca2+ level or require the entry of extracellular Ca2+. Photolysis of caged IP3 demonstrated that an increase in IP3 and a subsequent rise in Ca2+ do not produce desensitization of either the IP3 or 5-HT1c peak current responses. Furthermore, in oocytes coinjected with the low molecular weight RNA and a transcript from the rat M1 acetylcholine receptor, the M1 current response was greatly attenuated. Our data suggest that the proteins involved in attenuation of the M1 current response and desensitization of the 5-HT1c current response may be the same.
Insights
A low molecular weight RNA fraction from rat brain mRNA encodes proteins that desensitize serotonin 5-HT1c and M1 acetylcholine receptors in Xenopus oocytes. This desensitization is independent of intracellular calcium levels.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Signaling
Background:
- Serotonin 5-HT1c and acetylcholine M1 receptors activate phosphoinositidase, leading to increased IP3 and diacylglycerol.
- In Xenopus oocytes, IP3-mediated Ca2+ release opens Ca2+-gated Cl-channels, generating a measurable current response.
Purpose of the Study:
- To investigate the molecular mechanisms underlying receptor desensitization.
- To identify potential factors encoded by mRNA that modulate G protein-coupled receptor signaling.
Main Methods:
- Expression of mouse serotonin 5-HT1c receptor and rat M1 acetylcholine receptor in Xenopus oocytes via mRNA injection.
- Application of agonist pulses and measurement of oocyte current responses.
- Coinjection with low molecular weight rat brain mRNA fractions.
- Experiments using fura-2 and Ca2+-free superfusate to assess the role of intracellular and extracellular calcium.
- Photolysis of caged IP3 to probe IP3 signaling pathway.
Main Results:
- Oocytes expressing the 5-HT1c receptor showed a potentiated second current response compared to the first.
- Coinjection with low molecular weight RNA significantly reduced the second current response to ~50% of the first.
- Desensitization was not dependent on sustained intracellular Ca2+ or extracellular Ca2+ entry.
- Photolysis of caged IP3 did not induce desensitization of peak current responses.
- The M1 acetylcholine receptor current response was attenuated in oocytes coinjected with the low molecular weight RNA.
Conclusions:
- A low molecular weight RNA fraction from rat brain encodes protein(s) responsible for desensitizing the serotonin 5-HT1c receptor response.
- The desensitization mechanism does not involve sustained intracellular Ca2+ elevation or extracellular Ca2+ influx.
- The same protein(s) may be involved in both 5-HT1c receptor desensitization and M1 acetylcholine receptor response attenuation.