L-type Ca2+ channels regulate gene transcription, neuronal excitability and multiple forms of synaptic plasticity. Our long term interest is to determine the molecular mechanisms that regulate the L-type channel Cav1.2 (e.g., Science 293, 98; Science 293, 2205; PNAS Neuron 78, 483), which is the most prevalent L-type channel in brain and heart. We recently found that ?-actinin binds directly to the IQ motif of the centra pore-forming Cav1.2 subunit ?11.2 and augments its surface localization (Neuron 78, 483). We just identified three point mutations in the IQ motif that individually impair ?-actinin binding.Our preliminary electrophysiological and surface labeling data now suggest that impairing ?-actinin binding to the IQ motif decreases surface expression and, unexpectedly, also channel open probability (Po).
Aim 1 is the very first comprehensive analysis of trafficking kinetics of WT and point mutated Cav1.2 through the ER-Golgi-TGN secretory pathway and endocytic recycling and degradation pathway by surface biotinylation, N-glycosylation analysis and colocalization with respective markers like BiP and Rab5.
Aim 2 is to determine and compare current density, gating currents, and single channel currents to test whether the ?-actinin binding - deficient point mutations of Cav1.2 have decreased Po. Our structural NMR analysis is guiding charge reversal experiments for unequivocal (!) assignment of deficits in surface expression and Po in the Cav1.2 mutants to loss of ?-actinin binding. We recently discovered that LTP induced by the physiological Prolonged Theta-rhythm Tetanus (PTT, here 5Hz/180s; PTT-LTP) absolutely requires Cav1.2 but not NMDAR function.
Aim 3 will test our hypothesis that activity-induced alteration in postsynaptic Cav1.2 differentially affects PTT-LTP in young and old rats. Cav1.2 channel activity is strongly increased in aged rats (e.g., Science 272, 1017). The L-type channel inhibitor nimodipine impressively improves learning capabilities of aged rodents (e.g., Science 243, 809). Hence, an increase in Cav1.2 channel activity is thought to contribute to the etiology of senile symptoms and Alzheimer's disease, likely in part by impairing synaptic function. In fact we observed both, an increase in Cav1.2 surface expression and ?-actinin association in old vs. adult rat hippocampus. Thus our work on the functional interplay of Cav1.2 with ?-actinin and calmodulin is of high significance for understanding these aging-related neurological diseases. On a broader perspective it is of physiological importance as it will define important aspects that govern the functional availability of Cav1.2 with its manifold functions in neurons and beyond.
This project is to investigate the role of the physical interaction between the structural protein alpha-actinin and the Ca2+ channel Cav1.2, which is implicated in neurological and mental diseases such as dementia during aging, Alzheimer's disease, stroke, posttraumatic stress disorder, and autism. Alpha-actinin stabilizes Cav1.2 at the nerve cell surface. Prolonged Ca2+ influx leads to displacement of alpha-actinin. This event triggers internalization of Cav1.2 into the cell interior, which is important for curbing influx of Ca2+ into nerve cells, with too much Ca2+ influx causing neuronal damage leading to AD and other aging-related neurological and mental disorders. We will define the precise functional interplay between Cav1.2 and alpha-actinin to better understand surface targeting of Cav1.2 in health and aging-related diseases.
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