AKAP79 Anchoring in L-type Calcium Channel Regulation

描述(由申请人提供)： CAMP依赖的蛋白激酶使α1亚基C末端的单个丝氨酸残基上的L型钙通道磷酸化，从而增加去极化激活的通道数量。在肌肉中，PKA对LTCC的作用依赖于它与A激酶锚定蛋白(AKAP)的结合。在神经元中，LTCC定位于与AKAP79相似的亚细胞位置，AKAP不仅针对其底物的PKA，而且还针对磷酸酶钙调神经磷酸酶(CaN)。拟议研究的目标是确定PKA和是否可以共同影响LTCC的活动，从而需要AKAP79锚定。我将破坏PKA和CAN的AKAP79锚定，并通过使用全细胞电生理学测量LTCC峰值电流的变化来研究这如何影响这些酶调节通道的能力。其次，我将利用荧光共振能量转移来测量通道-AKAP-PKA/-CaN复合体成员之间分子间亲和力的变化速度，并通过同时记录钙通道电流来将这些变化与LTCC调节相关联。

DESCRIPTION (provided by applicant): CAMP-dependent kinase (PKA) phosphorylates the L-type calcium channel (LTCC) at a single serine residue in the C-terminus of the alpha1 subunit and in doing so increases the number of channels that will be activated by depolarization. In muscle PKA action on the LTCC is dependent upon its binding to A kinase anchoring proteins (AKAPs). In neurons LTCCs localize to similar subcellular locations as AKAP79, an AKAP that not only targets PKA to its substrates, but also the phosphatase calcineurin (CaN). The goal of the proposed research is to determine whether PKA and CaN together influence the activity of the LTCC in a manner that requires AKAP79 anchoring. I will disrupt AKAP79 anchoring of PKA and CaN and study how this affects the ability of these enzymes to regulate the channel by measuring changes in LTCC peak current using whole cell electrophysiology. Secondly, I will measure the rates of changes in intermolecular proximity between members of the channel-AKAP-PKA/-CaN complex using fluorescence resonance energy transfer and correlate these changes with LTCC regulation by simultaneously recording calcium channel currents.