During the past funding period, we have discovered a number of details of the mechanisms underlying acute ethanol inhibition of voltage-gated calcium channels (VGCC)in PC12 cells, including the involvement of identified G-proteins. We have also discovered that differentiation with nerve growth factor (NGF), as well as chronic exposure to ethanol, significantly decreases acute ethanol inhibition. We plan to focus the next phase of our research on the mechanisms underlying the inhibition of Ga channels. This work, which will use a combination of molecular biology and biophysics, will be performed on three types of preparation, each contributing particular advantages: 1) intact PG12 cells, where we will further elucidate the mechanisms of ethanol action on VGCC; 2) the intact oocyte expression system, where we can study selected populations of channels and components, such as G-proteins, in a cellular environment; and 3)internally perfused oocytes, in which we can manipulate both the interior and exterior environment, maintaining a natural membrane and underlying cytoskeleton. At each level, we will study channels from undifferentiated cells, differentiated cells, and cells chronically exposed to alcohol, allowing us to gain an understanding for the basis of differences in acute sensitivity.
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