Shigehiro Nakajima - US grants

One of the goals of this project is to understand the mechanisms by which contraction is initiated and controlled by potential changes of the membranous system of skeletal muscle. Electrical and membranous properties of the transverse tubular system (T-system) and sarcoplasmic reticulum will be explored by using optical methods (fluorescence dye absorption and external fluorescence method) as well as by the conventional electrophysiological techniques. We would particularly like to know details about the radial conduction of action potential through the T-system and about the membrane potential changes of sarcoplasmic reticulum during excitation-contraction coupling. Another goal of this project is to investigate the mechanism of fatigue in synaptic transmission by using the squid stellate ganglion.

brain cell; biophysics; tissue /cell culture;

There are many transmitter substances which regulate the activity of nerve cells of the central nervous system. The objective of this proposal is to elucidate the mechanisms by which these transmitter substances activate intracellular messenger processes that lead to the modulation of excitability through their action on potassium channel conductance properties. Recent studies have shown that excitability of neurons is dependent on potassium channels which are, in turn, regulated by G-proteins called Gi and Go, of which there are several subtypes. The specific aim of this investigation is to identify which of the G-protein subtypes are related to neurotransmitter action (acetylcholine and somatostatin). Molecular biological techniques will be used to produce specific mutations to selectively inactivate G-proteins and thereby determine which of them is directly affected by the neurotransmitters to act on potassium conductance of the cell. For this purpose mutated alpha subunits will be inserted into AtT-20 cells, a pituitary tumor cell line which contain G-protein regulated potassium channels. The effect of the mutants can then be ascertained on potassium channel function.

This grant project investigates the properties of a non- selective ion channel (channels are functionally important proteins located in the cell membrane). The non-selective channel plays an important part in slow excitation of brain cells induced by brain chemicals. The overall summation of the excitatory state of each of the brain cells in each specialized nucleus (region) of the brain determines the tone of the brain. In spite of its widespread nature and its functional importance, the non-selective channel has long been ignored, and little is known about its properties. To investigate this channel in brain neurons has been difficult because of the lack of a suitable cell type. Recently, the laboratory of the principal investigator has found that cultured neurons from the ventral tegmental area (a brain region playing an important part in the pathogenesis of schizophrenia) are an excellent cell type for this purpose, since this channel is consistently activated by four excitayory chemicals: (1) neurotensin, (2) neurokinin B, (3) metabotropic glutamate agonist, (4) and muscarine Thus, it is now feasible to conduct a systematic investigation of this channel. The specific projects examine: (1) whether each one of these brain chemicals acts on its own receptor, (2) the permeability of the channel to various ions, (3) the signal transduction mechanism (details of the events leading to a slow excitation of the neuron). (4) the pharmacology of this channel, and (5) the single channel properties of this channel. Preliminary results suggest that this non- selective channel has unusual characteristics: (1) It is different from a Ca-activated non-specific cation channel. (2) The channel activiation is independent of G proteins (an important protein for many types of signal transduction). (3) Yet, it does not seem to be a ligand- gated channel (usual channel for very quick excitation), since the latency of activation is very long.

The objective of this research proposal is to study the regulatory mechanisms of brain neurons that are implicated in major mental disorders. The proposed experiments will be done mainly by using primary cultured dopaminergic (DA) neurons from the substantia nigra and the ventral tegmental area of the rat brain. The methods will be electrophysiological techniques combined with molecular biological methods. The first project is concerned with excitation of these DA neurons. Neurotensin produces a slow excitation of DA neurons by activating a non-selective cation channel. The analysis of the ionic mechanism has revealed that this non-selective channel is different from the well-known non-selective ion channels such as the CAN channels (the calcium-activated non-specific cation channels) or the cyclic nucleotide-gated channels. The proposed experiments will shed light on the signal transduction mechanisms of the neurotensin-induced activation of this non-selective channel. The role played by G proteins will be critically evaluated, and the identity of the G protein will be determine. The other project deals with a slow inhibition of the DA neurons by dopamine itself. This self-inhibition results from an activation of an inward rectifier K+ channel by the G protein betagamma- subunits. The project will investigate the mechanisms of the interaction of the betagamma-subunits and the inward rectifier molecule. It is proposed to find a convenient way to transfect primary cultured DA neurons with plasmid cDNA. Then the DA neurons (also HEK293 cells) will be transfected with CDNAs encoding the G protein beta-subunit and the gamma-subunit. The effects of site-directed mutations of the beta- subunit will be evaluated. The investigation will show which regions of the beta-subunit molecule are responsible for the association with and activation of the inward rectifier molecule.