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Activation of 5-HT3 receptors expressed in Xenopus oocytes does not increase cytoplasmic Ca2+ levels
1Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, N.C. 27709, USA.
Abstract:
The wildtype 5-HT3 receptor was expressed in Xenopus oocytes from a cloned cDNA. Two-electrode voltage-clamp and fura-2 techniques were used simultaneously to monitor the current induced by the activation of the 5-HT3 receptor and the cytoplasmic Ca2+ concentration ([Ca2+]i). The application of serotonin (5-HT; 50 microM) in a physiological bathing solution containing Ca2+ (1.8 mM) elicited inward currents of up to approximately 1.3 microA at a potential of -90 mV. There was little or no detectable change in [Ca2+]i. In experiments on oocytes bathing in a nominally Ca(2+)-free medium, the injection of (2,4,5)IP3, an analog of inositol 1,4,5-trisphosphate ((1,4,5)IP3), produced a large increase in [Ca2+]i levels due to the liberation of Ca2+ from intracellular stores. This response was followed by a sustained elevation of [Ca2+]i upon restoring extracellular Ca2+ (i.e. capacitative Ca2+ entry). Furthermore, when calcium-permeable heteromeric (NR1/NR2A) N-methyl-D-aspartate (NMDA) receptors were expressed in oocytes, the activation of these receptors led to a large increase in [Ca2+]i in a Ca(2+)-containing external medium. It is concluded that wildtype 5-HT3 receptors expressed in Xenopus oocytes and studied under conditions used here show little or no permeability for Ca2+.
Insights
Wildtype serotonin 3 (5-HT3) receptors in Xenopus oocytes show minimal calcium permeability. Experiments using two-electrode voltage-clamp and fura-2 confirmed little to no influx of Ca2+ upon receptor activation.
Area of Science:
- Neuroscience
- Molecular Biology
- Ion Channel Physiology
Background:
- The serotonin 3 (5-HT3) receptor is a ligand-gated ion channel implicated in various physiological processes.
- Understanding the ion permeability of 5-HT3 receptors is crucial for elucidating their role in cellular signaling.
Purpose of the Study:
- To investigate the calcium (Ca2+) permeability of wildtype 5-HT3 receptors expressed in Xenopus oocytes.
Main Methods:
- Expression of cloned wildtype 5-HT3 receptor cDNA in Xenopus oocytes.
- Simultaneous two-electrode voltage-clamp and fura-2 measurements to monitor receptor currents and intracellular Ca2+ ([Ca2+]i).
- Application of serotonin (5-HT) and other stimuli to assess Ca2+ influx and release.
Main Results:
- Serotonin (5-HT) application induced inward currents but little to no detectable change in [Ca2+]i.
- Inositol 1,4,5-trisphosphate ((1,4,5)IP3) injection caused Ca2+ release from intracellular stores, followed by capacitative Ca2+ entry.
- Activation of calcium-permeable N-methyl-D-aspartate (NMDA) receptors led to significant [Ca2+]i increases, serving as a positive control.
Conclusions:
- Wildtype 5-HT3 receptors expressed in Xenopus oocytes exhibit negligible permeability to Ca2+ under the experimental conditions.
- The study differentiates 5-HT3 receptor function from other Ca2+-permeable ion channels like NMDA receptors.