The long-term objective of this proposal is to determine how voltage-activated ion channels are trafficked and localized in neuronal dendrites, where the channels play important roles in regulating excitability and integration of synaptic inputs. In particular, I will examine the trafficking of the hyperpolarization-activated cyclic nucleotide-gated HCN1 channel, which is targeted to distal dendrites of pyramidal neurons, and whose surface expression is regulated by patterns of activity that induce long-term plasticity or seizures. 1. Which regions of HCN1mediate targeting to distal dendrites? In preliminary results I found a region of the C-terminus of HCN1 that is necessary for its localization to distal dendrites. This same region of HCN1 is critical for the binding of TRIP8b, a proposed HCN1 trafficking protein1. To test if this region is sufficient for distal dendritic targeting, sequences derived from the C-terminus of HCN1 will be added to the intracellular C-terminus of glycoprotein differentiation cluster 8 (CD8), a T-cell membrane protein not targeted to distal dendrites. These constructs will be expressed with lentivirus in mature hippocampal CA1 pyramidal neurons to define a minimal HCN1-derived dendritic-targeting domain. 2. Can the HCN1 targeting domain be developed as a tool to study dendritic integration? Once an HCN1 sequence has been identified that is sufficient for targeting CD8, I will develop novel localized regulators of distal dendritic function by fusing the trafficking sequence to foreign proteins, such as the G- protein coupled Drosophila allatostatin receptor to enable ligand-dependent inhibition of distal dendrites, or channelrhodopsin to provide light-activated excitation of distal dendrites. Such tools will enable the study of the role of distal dendrites in brain circuitry and behavior. 3. Is the trafficking of HCN1 influenced by synaptic activity? There is mounting evidence that the surface expression of HCN1 changes in response to seizures or more physiological patterns of synaptic activity used to induce LTP. However, altered HCN1 trafficking has not been directly demonstrated. The motion of GFP-tagged HCN1 during the induction of LTP and seizure-like activity will be monitored to detect changes in channel trafficking. The contribution of HCN1 trafficking proteins will also be examined. Relevance to public health: Changes in the levels of the HCN1 ion channel in response to seizures is thought to contribute to the development of epilepsy. Findings from this research will hopefully contribute to the understanding of how the HCN1 channel helps regulate neural excitability and how seizures regulate channel trafficking and expression. Such results may aid in the development of new disease treatments.
|Piskorowski, Rebecca; Santoro, Bina; Siegelbaum, Steven A (2011) TRIP8b splice forms act in concert to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons. Neuron 70:495-509|
|Santoro, Bina; Hu, Lei; Liu, Haiying et al. (2011) TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites. J Neurosci 31:4074-86|