ABSTRACT
Most G protein-coupled receptors (GPCRs) do not generate membrane currents in response to ligand-receptor binding (LRB). Here, we describe a novel technique using endocytosis as a bioassay that can detect activation of a GPCR in a way analogous to patch-clamp recording of an ion channel in a living cell. The confocal imaging technique, termed FM endocytosis imaging (FEI), can record ligand-GPCR binding with high temporal (second) and spatial (micrometer) resolution. LRB leads to internalization of an endocytic vesicle, which can be labeled by a styryl FM dye and visualized as a fluorescent spot. Distinct from the green fluorescence protein-labeling method, FEI can detect LRB endocytosis mediated by essentially any receptors (GPCRs or receptors of tyrosine kinase) in a native cell/cell line. Three modified versions of FEI permit promising applications in functional GPCR studies and drug screening in living cells: 1) LRB can be recorded in "real time" (time scale of seconds); 2) internalized vesicles mediated by different GPCRs can be discriminated by different colors; and 3) a high throughput method can screen ligands of a specific GPCR.
Subject(s)
Endocytosis , Ganglia, Spinal/metabolism , Ligands , Microscopy, Confocal/methods , Molecular Imaging/methods , Neurons/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Fluorescent Dyes/metabolism , Ganglia, Spinal/cytology , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Pyridinium Compounds/metabolism , Quaternary Ammonium Compounds/metabolism , Rats , Rats, Wistar , Receptors, Adrenergic/metabolism , Receptors, Cholinergic/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, GABA-B/metabolism , Receptors, Nerve Growth Factor/metabolism , Receptors, Purinergic P2Y1/metabolism , Receptors, Serotonin/metabolism , Recombinant Fusion Proteins/metabolism , Time Factors , TransfectionABSTRACT
The somata of dorsal root ganglion (DRG) neurons release neurotransmitters and neuropeptides. In addition to the conventional Ca2+-dependent secretion (CDS), Ca2+-independent but voltage-dependent secretion (CIVDS) also occurs in the somata of DRG neurons. Electrical stimulation induces both CDS and CIVDS, which differ in size and are coupled with different types of endocytosis contributed by CIVDS and CDS, respectively. However, it is unclear whether they use a common vesicle pool, so we investigated the relationship between the vesicle pools of CDS and CIVDS. Membrane capacitance recording and photolysis of a caged-Ca2+ compound showed that, in low external Ca2+ solutions, the depolarization-induced exocytosis contained two (fast and slow) phases, which were contributed by CIVDS and CDS, respectively. Depletion of the CDS readily releasable pool using photolysis did not affect the CIVDS. When the CIVDS and CDS vesicle pools were depleted by electrical stimulation, the pools had different sizes. Their kinetics of exocytosis-coupled endocytosis were also different. Thus, CIVDS and CDS used different vesicle pools in DRG neurons.
Subject(s)
Exocytosis/physiology , Ganglia, Spinal/physiology , Neurons/physiology , Animals , Calcium/metabolism , Cells, Cultured , Electric Capacitance , Endocytosis/physiology , Female , Male , Membrane Potentials/physiology , Neurotransmitter Agents/physiology , Rats , Rats, Wistar , Secretory Vesicles/physiology , Synaptic Transmission/physiologyABSTRACT
Neurotransmitter release normally requires calcium triggering. However, the somata of dorsal root ganglion (DRG) neurons possess a calcium-independent but voltage-dependent secretion (CIVDS) in addition to the classic calcium-dependent secretion (CDS). Here, we investigated the physiological role of CIVDS and the contributions of CIVDS and CDS induced by action potentials (APs) in DRG soma. Using membrane capacitance measurements, caged calcium photolysis, and membrane capacitance kinetics analysis, we demonstrated that AP-induced secretion had both CIVDS and CDS components. Following physiological stimuli, the dominant component of AP-induced secretion was either CIVDS for spontaneous firing or CDS for high-intensity stimuli. AP frequency modulates CDS-coupled exocytosis and CIVDS-coupled endocytosis but not CIVDS-coupled exocytosis and CDS-coupled endocytosis. Finally, CIVDS did not contribute to excitatory postsynaptic currents induced by APs in DRG presynaptic terminals in the spinal cord. Thus, CIVDS is probably an essential physiological component of AP-induced secretion in the soma. These findings bring novel insights into primary sensory processes in DRG neurons.
