REGULATION OF T-TYPE CALCIUM CHANNEL TRAFFICKING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. T-type Ca2+ channels are an important group of voltage-gated ion channels that regulate the electrophysiological properties of neurons as well as various Ca2+-dependent cellular processes. T-type Ca2+ channels regulate the action potential waveform and the temporal pattern of repetitive firing of nerve cells and therefore play a critical role in defining the electrophysiological phenotype of neurons. Activation of T-type Ca2+ channels may also play a critical role in nociception or sensory pain transmission. For example, elimination of T-type Ca2+ channels results in a significant decrease in pain-evoked responses. Although the role of T-type calcium channels in sensory perception and nociception is well established we have very little understanding of the molecular and cellular mechanisms that regulate T-type channel expression in a sensory neuron lifetime. We have previously determined that the expression of T-type Ca2+ channels in sensory neurons is developmentally regulated. At arly stages of development, T-type Ca2+ channel expression is very low and is increased several fold by birth. This proposal is designed to test the hypothesis that neurotrophic factors regulate the expression of T-type Ca2+ channels in developing sensory neurons. To test this hypothesis we will use the chicken dorsal root ganglia (DRG) neurons as a model. These are sensory neurons that transmit a variety of sensory stimuli including pain sensations to the central nervous system. Based on their electrophysiological and morphological properties, DRG neurons can be divided into C-type and A-type neurons. Pain transmitting neurons appear to be C-type. These cells are characterized by their small cell bodies and prolonged action potentials. Pain-transmitting DRG neurons also appear to label for lectin agglutinin. This will allow us to stain only C-type, pain sensitive DRG neurons in our studies. First, we will establish the developmental expression of Ttype Ca2+ channels in isolated DRG neurons. Neurons will be isolated from chicken embryos at embryonic day (E) 6, E10, E15 and E20. T-type Ca2+ channel expression will be determined by voltage-clamp recordings of lectin-labeled DRG neurons. The ability of different neuronal types of DRG neurons to express T-type Ca2+ channels will be investigated by comparing the level of T-type Ca2+ channel expression in lectin-positive and lectin-negative neurons. Second, we will investigate whether exposure of DRG neurons to various trophic factors regulate the expression of T-type Ca2+ channels. The survival and differentiation of DRG neurons depends on the neurotrophin nerve growth factor (or NGF). Other factors like the cytokines ciliary neurotrophic factor (or CNTF) or leukemia inhibitory factor (or LIF) also may regulate the survival and differentiation of DRG neurons. Therefore, we will test whether in the presence NGF, CNTF or LIF, DRG neurons are more likely to express T-type Ca2+ channels. Neurons will be isolated at E6 or at later stages of development and culture for 24 to 72 hr in the presence of 10 ng/mL of NGF, CNTF or LIF. Channel expression will be assessed by electrophysiological recordings in lectin-labeled neurons.