TY - JOUR
T1 - Distinct subcellular targeting of fluorescent nicotinic α3β4 and serotoninergic 5-HT3A receptors in hippocampal neurons
AU - Grailhe, Régis
AU - De Carvalho, Lia Prado
AU - Paas, Yoav
AU - Le Poupon, Chantal
AU - Soudant, Martine
AU - Bregestovski, Piotr
AU - Changeux, Jean Pierre
AU - Corringer, Pierre Jean
PY - 2004/2
Y1 - 2004/2
N2 - The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of α3β4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric α3CFP-β4 and YFP-α3β4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT 3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, α3β4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of α3β4-nACh receptors and the predominant expression of the 5-HT 3AR in dendritic surface loci.
AB - The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of α3β4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric α3CFP-β4 and YFP-α3β4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT 3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, α3β4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of α3β4-nACh receptors and the predominant expression of the 5-HT 3AR in dendritic surface loci.
KW - GFP
KW - Nicotinic
KW - Receptors
KW - Serotoninergic
KW - Targeting
UR - http://www.scopus.com/inward/record.url?scp=1542359609&partnerID=8YFLogxK
U2 - 10.1111/j.1460-9568.2004.03153.x
DO - 10.1111/j.1460-9568.2004.03153.x
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C2 - 15009132
SN - 0953-816X
VL - 19
SP - 855
EP - 862
JO - European Journal of Neuroscience
JF - European Journal of Neuroscience
IS - 4
ER -