Function of Ca2+ channel beta4 subunit in the nucleus

In the nervous system voltage-gated calcium (Ca 2+) channels play important roles in synaptic transmission, in synaptic plasticity, and in the activity-dependent regulation of gene expression during development and learning. Ca2+ entering through voltage-gated Ca2+ channels can regulate transcription either via cytoplasmic signaling cascades or locally in the nucleus. Emerging evidence indicates a new route of Ca2+ channel-dependent gene regulation, involving the translocation of channel fragments into the nucleus. Recently, we and others observed targeting of the Ca2+ channel ß 4 subunit into nuclei of various cell types. Therefore we hypothesize that, in addition to its function in the Ca2+ channel complex, ß 4 may function in the regulation of gene expression in excitable cells. If this is the case, we expect that ß 4 nuclear targeting is specific, that it is regulated during development and/or activity, that ß 4 interacts directly or indirectly with transcriptional regulatory elements, and that ß 4 nuclear targeting regulates the expression of endogenous genes. Thus, the overall goal of this research project is to determine the function of the auxiliary Ca2+ channel ß 4 subunit in the nucleus of excitable cells. This shall be accomplished by (1) studying the mechanism of ß 4 nuclear targeting, (2) characterizing the regulation of ß 4 nuclear targeting in excitable cells, and (3) examining the possible involvement of ß 4 in gene regulation in neurons. Experimentally the prediction of our hypothesis shall be examined by expressing and analyzing tagged ß subunits in culture models of excitable cells. The specificity and the regulation of ß 4 nuclear targeting as well as the nature of nuclear import/export signals will be studied using heterologous ß subunits, chimeras and mutants expressed with and without a 1 subunits in dysgenic muscle cells. FRAP and photoactivation analysis of GFP- and Dendra- ß 4 constructs will be used to study the mechanism of ß 4 nuclear targeting in nerve and muscle cells. Gene array analysis and immunoprecipitation combined with mass spectroscopy will be used to identify ß 4 -regulated genes and binding partners of ß 4 in the nucleus of nerve cells. Addressing the question of the nuclear function of ß 4 will significantly contribute to an emerging field of ion channel research and can be expected to reveal fundamentally new mechanisms of gene regulation via voltage-gated Ca2+ channels. In neurons this hitherto unappreciated function of ß 4 may contribute to cellular processes underlying learning and memory and may be causally related to diseases like absence epilepsy and ataxia in mice and patients with loss-of-function mutations of the Ca2+ channel ß 4 subunit.

In the nervous system voltage-gated calcium (Ca 2+) channels play important roles in synaptic transmission, in synaptic plasticity, and in the activity-dependent regulation of gene expression during development and learning. Ca2+ entering through voltage-gated Ca2+ channels can regulate transcription either via cytoplasmic signaling cascades or locally in the nucleus. Emerging evidence indicates a new route of Ca2+ channel-dependent gene regulation, involving the translocation of channel fragments into the nucleus. Recently, we and others observed targeting of the Ca2+ channel ß 4 subunit into nuclei of various cell types. Therefore we hypothesize that, in addition to its function in the Ca2+ channel complex, ß 4 may function in the regulation of gene expression in excitable cells. If this is the case, we expect that ß 4 nuclear targeting is specific, that it is regulated during development and/or activity, that ß 4 interacts directly or indirectly with transcriptional regulatory elements, and that ß 4 nuclear targeting regulates the expression of endogenous genes. Thus, the overall goal of this research project is to determine the function of the auxiliary Ca2+ channel ß 4 subunit in the nucleus of excitable cells. This shall be accomplished by (1) studying the mechanism of ß 4 nuclear targeting, (2) characterizing the regulation of ß 4 nuclear targeting in excitable cells, and (3) examining the possible involvement of ß 4 in gene regulation in neurons. Experimentally the prediction of our hypothesis shall be examined by expressing and analyzing tagged ß subunits in culture models of excitable cells. The specificity and the regulation of ß 4 nuclear targeting as well as the nature of nuclear import/export signals will be studied using heterologous ß subunits, chimeras and mutants expressed with and without a 1 subunits in dysgenic muscle cells. FRAP and photoactivation analysis of GFP- and Dendra-ß 4 constructs will be used to study the mechanism of ß 4 nuclear targeting in nerve and muscle cells. Gene array analysis and immunoprecipitation combined with mass spectroscopy will be used to identify ß 4 -regulated genes and binding partners of ß 4 in the nucleus of nerve cells. Addressing the question of the nuclear function of ß 4 will significantly contribute to an emerging field of ion channel research and can be expected to reveal fundamentally new mechanisms of gene regulation via voltage-gated Ca2+ channels. In neurons this hitherto unappreciated function of ß 4 may contribute to cellular processes underlying learning and memory and may be causally related to diseases like absence epilepsy and ataxia in mice and patients with loss-of-function mutations of the Ca2+ channel ß 4 subunit.