Voltage-dependent channels on the dendrites of motoneurons provide additional current that amplifies or dampens synaptic current en route to the soma. The specific consequences will depend on the density and distribution of the voltage-dependent channels. HCN channels generate a positive inward current in response to hyperpolarization. HCN channels are responsible for a resonance phenomenon in motoneurons where inputs of certain frequencies are preferentially amplified. Modelling studies indicate that this resonance behaviour only occurs if HCN channels are uniformly distributed on the dendritic tree. However, the distribution of HCN channels on the dendrites of motoneurons is unknown. Furthermore, current techniques for measuring channel density on dendrites suffer from methodological limitations that prevent sampling on a scale necessary to map the distribution of voltage-dependent channels across the dendritic tree. The goal of the present study is to develop a high throughput method for measuring the membrane-associated density of voltage-dependent channels using immunohistochemical, confocal and three-dimensional image analysis techniques. Secondly, the proximal to distal distribution of membrane-associated channels formed by the HCN1-subunit on the dendritic trees of trapezius motoneurons in the adult feline will be compared. Antidromically identified motoneurons innervating the trapezius muscle were intracellularly stained in order to visualize the entire dendritic tree. HCN1-subunit containing channels were labeled with a specific antibody. Dendritic segments (n=27 to 92) of ten trapezius motoneurons at different distances from the soma were acquired using confocal microscopy and rendered into a three-dimensional volume. The perimeter of the intracellular stain was used to define the membrane-cytoplasmic border. Analysis of the HCN1 immunoreactivitywas constrained to this perimeter. This technique provides a means of extracting membrane-associated HCN1 labeling and therefore the functional distribution of HCN1 channels. The density of membrane-associated HCN1 immunoreactvity across the dendritic tree was either uniform or increased with distance from the soma among the ten trapezius motoneurons. The increase in HCN1 density with distance from the soma was inversely related to the density of HCN1 on the soma and proximal dendrites. Lastly, the change in HCN1 density with distance from the soma was inversely related to the total input conductance of the motoneuron. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-08-01 13:59:32.403

Photoreceptors are highly compartmentalized neurons in the retina, and they function by detecting light and initiating signaling through the visual network. The photoreceptor contains several compartments including the outer segment (OS) which is a sensory cilium for detecting photons and the inner segment (IS) that carries out important modulatory functions via its resident channels and transporters. Those proteins are membrane proteins that function together to shape electrical properties of the cell membrane during both rest and active states. Therefore it is essential to maintain proper function of the membrane proteins in the IS. One important way to regulate the function of a membrane protein is via controlling its trafficking to ensure a proper amount of the protein in the proper cellular compartment. To date, little is known about how IS membrane protein trafficking is controlled in photoreceptors. In this study, our goal is to understand those mechanisms using cell biology and biochemistry approaches. To achieve the goal, we investigated trafficking of two unrelated IS resident proteins: the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) that mediates a feedback current in photoreceptors, and the sodium potassium ATPase (NKA) which maintains the basic electrochemical property of the cell. In order to study trafficking of HCN1, we first investigated the dependence of HCN1 trafficking in photoreceptors on TRIP8b, an accessory subunit that influences trafficking of HCN1 in hippocampal neurons. By studying TRIP8b knockout mice we found that TRIP8b is dispensable for HCN1 trafficking in photoreceptors but required for maintaining the maximal expression level of HCN1. Since we revealed that HCN1 trafficking can be regulated in a cell-type specific manner, we subsequently focused on the amino acid sequence of HCN1 to identify novel trafficking signals that function in photoreceptors. By examining localization of a series of HCN1 mutants in transgenic Xenopus photoreceptors, we discovered a di-arginine ER retention motif and a leucine-based ER export motif. These two sequence motifs must function together to maintain equilibrium of HCN1 level between the endomembrane system and the cell surface. The study of HCN1 uncovered a mechanism for the photoreceptor to control membrane protein trafficking via the early secretory pathways. To reveal additional trafficking machineries in photoreceptors, we investigated trafficking of NKA. We first tested for an interaction with ankyrin, an adaptor protein that regulates NKA trafficking in epithelial cells, and found these proteins do not co-localize in photoreceptors. We then aimed to identify novel trafficking signals by studying the trafficking behavior of two NKA isozymes: NKA-α 3 and NKA-α 4. When expressed in transgenic Xenopus photoreceptors, these two proteins localize to the IS and the OS respectively. By studying localization of multiple chimeras and truncation mutants, we found that the distinct localization pattern is due to a VxP OS/ciliary targeting motif present in NKA-α 4. Since NKA-α 4 is naturally expressed in the ciliary compartment of the sperm, our finding in the photoreceptor suggests a mechanism for NKA-alpha 4 trafficking in its native environment. Overall, our studies of HCN1 and NKA together provide new insights into controlling membrane protein trafficking in photoreceptors and help establish the basics for future therapeutic intervention targeting trafficking pathways that are linked to about one third of proteins reported in retinal diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21692号 / 医科博第96号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 渡邉 大, 教授 伊佐 正, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM