| Many neurotransmitters, hormones and sensory stimuli exert their biological functions through transmembrane receptors which couple to heterotrimeric guanine nucleotide binding protein (G protein) and normally have 7 transmembrane domains called G-protein coupled receptor (GPCR). G proteins work as transducers which convert extracellular signals into intracellular events. Voltage-gated calcium channels (VGCC) (P,Q and N types) and G-protein inwardly rectifying potassium channels (GIRK) are important for modulation of neuronal excitability and synaptic communication. They also belong to the end-targets of GPCRs. These modulations are mediated by G protein betagamma subunits. Combining electrophysiological recordings and fluorescence resonance energy transfer (FRET), I characterized different regions derived from Gbeta2 subunit for interaction with P/Q type calcium channels and GIRK channels. Interestingly, different parts of the Gbeta2 subunit can either induce or inhibit G protein modulation of the examined ion channels. In particular, peptides derived from the Gbeta2 N-terminus inhibit G protein modulation, whereas peptides derived from Gbeta2 C-terminus induced channel modulation.; In a second series of studies, I developed light activated probes which could control neuronal excitability. First, I demonstrated that vertebrate rhodopsin can couples to GIRK channels and voltage gated Ca2+ channels through the Gi/o pathway. When expressed in hippocampal neurons, light activation of rhodopsin reduces neuronal firing and modulates synaptic transmission. In contrast, to excite neurons, I applied the green algae channelrhodospin 2 (ChR2), which could generate inward sodium current in HEK293 cells in response to light. Moreover, when ChR2 was expressed in hippocampal neurons, light activation ChR2 can depolarizes neurons to induce action potentials and presynaptic neuronal transmitter release. To further characterize the use of these two light switches in neuronal regulation, I used embryonic chick spinal cords as a model. Electroporated with vertebrate rhodopsin, turning on light could reduce the spontaneous firing and synchronize the bursting activity of spinal cord; on the other hand, light could increase spontaneous activity of spinal cord which was electroporated with ChR2. Thus, with these two probes, I could either increase or decrease neuronal activity with light in a fast and non-invasive way. |
