The overall goal of this project is to gain a clearer picture of how voltage-gated calcium channels link membrane excitation to rises in intracellular Ca2+ and vital cellular responses. The specific focus is on the alpha1A subunit, which generates P/Q type channels, the dominant pathway for Ca2+ current in many CNS neuron cell bodies and nerve terminals. Deletion of alpha1A drastically changes the profile of pre- and postsynaptic Ca2+ entry and alters key properties of synaptic transmission; mutations in alpha1A can cause neurological diseases, including migraine, episodic ataxia, and spinocerebellar ataxia in human, and various forms of absence epilepsy in mice. Here one major aim is to use molecular strategies to shed light on the molecular relationship between Ca2+ channels and Ca2+ sensors for transmitter release and short term facilitation. Engineered versions of human or mouse alpha1A subunits will be introduced as transgenes driven by the endogenous mouse promoter. Among the modified alpha1A subunits will be one unable to support Ca2+ permeation but with cytoplasmic 0domains intact, and another fully able to permeate but lacking in domains thought to be necessary for interaction with the release machinery. In this way, distinctions will be made between possible roles of alpha1A subunits as providers of Ca2+ influx and a putative structural elements within the synapse. Similar approaches will be directed toward understanding familial hemiplegic migraine (FHM), which arises from points mutations in alpha1A. Previous biophysical studies of the FHM mutants expressed in oocytes or HEK293 cells have not yielded a clear picture of what Ca2+ channel characteristics cause the disease. However, it is expected that a more consistent pattern will emerge when these mutants are studies in a physiological setting within mammalian brain cells and their participation in Ca2+ homeostasis is taken into account. Regulation of pre- and postsynaptic Ca2+ transients and synaptic transmission will be examined to see how these are affected by the various modifications in alpha1A. This work may provide useful clues about the dominant pattern of inheritance of FHM and the intermittent features of the disease.
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