電位依存性イオンチャネル研究のフロンティア:ホジキン・ハックスレーから60年を経て
Frontiers of Voltage-gated Ion Channels: At 60 year after the discovery in Squid Giant Axon
S1-5-1-1
電位依存性ナトリウムチャネルとてんかん/自閉症
Voltage-gated sodium channel and epilepsy / autism

○山川和弘1
○Kazuhiro Yamakawa1
理化学研究所脳科学総合研究センター1
Laboratory for Neurogenetics, RIKEN Brain Science Institute1

Mutations of voltage-gated sodium channel genes have been reported in patients with epilepsies associated with autism. Inherited missense mutations of SCN1A gene encoding Nav1.1 have been described in patients with generalized epilepsy with febrile seizures plus (GEFS+) characterized by autosomal-dominant inheritance, febrile and afebrile seizures, and occasionally associates with psychiatric features such as panic disorder or Asperger syndrome. De novo loss of function mutations of SCN1A have been described in patients with Dravet syndrome (alternatively named as severe myoclonic epilepsy in infancy: SMEI) which is sporadic intractable epilepsy associated with severe mental decline, ataxia, and autistic feature. We reported that mouse with SCN1A nonsense mutation showed epileptic seizures and physiological dysfunction in first-spiking GABAergic neurons. In wild type mouse, Nav1.1 protein was dominantly expressed in axons and somata of parvalbumin-positive inhibitory interneurons. Additional data further suggest that functional impairments of parvalbumin-positive interneurons caused by Nav1.1 haploinsufficiency is the pathological basis of Dravet syndrome. Mutations of SCN2A gene encoding Nav1.2, although the expression pattern is largely distinct to Nav1.1, have also been described in a wide spectrum of epilepsies.We were the first to report a mutation of SCN2A in a patient with epilepsy, which was atypical GEFS+. Subsequently, multiple missense mutations in SCN2A have been described in benign familial neonatal-infantile seizures (BFNIS). We further found sporadic de novo mutations of SCN2A in patients with intractable epilepsy, severe mental decline, and some associate with autism. Recently, multiple large scale full-genome exome sequencing studies also described multiple de novo loss of function mutations of SCN1A and SCN2A in patients with autism. These observations indicate that mutations of these genes are responsible for autism in addition to epilepsy.
S1-5-1-2
チャネルの神経細胞内局在を制御する分子メカニズム
Mechanisms regulating precise localization of voltage-gated K+ channels in neurons

○御園生裕明1
○Hiroaki Misonou1
同志社大学 脳科学研究科1
Graduate School of Brain Science, Doshisha University1

The computational ability of nerve cells depends on the specific localization of ion channels in neuronal membrane. Most ion channels are targeted to either the axonal or somatodendritic compartments, where they are further localized to more discrete membrane sub-compartments, such as dendritic spines. However, the molecular mechanism which determines the sub-compartment specific localization of ion channels is largely unknown. Here, we focused on dendritic voltage-gated potassium channels, Kv2.1 and Kv4.2, to uncover the molecular basis of the specific localization of ion channels in dendrites. Kv2.1 is localized in the cell body and the proximal part of the dendrites, where it impacts the integration of synaptic potentials and centrally controls the overall excitability of neurons. In contrast, Kv4.2 is abundantly expressed in the distal dendrites, where it locally regulates the propagation and integration of synaptic potentials. It has been proposed that these channel proteins may be targeted to specific sub-compartments of dendrites by intracellular trafficking mechanisms, but direct evidence regarding how this might be accomplished in living neurons has been lacking. We hypothesized that dendritic Kv2.1 and Kv4.2 channels are sorted into distinct transport vesicles and selectively delivered by unique transport systems to sub-compartments of dendrites. To test this, We employed quantitative live-cell imaging of the channel subunits tagged with fluorescent proteins in cultured rat hippocampal neurons. Our results show that Kv2.1 and Kv4.2 are sorted in unique populations of transport vesicles, differentially targeted at the level of vesicular trafficking, and transported by specific molecular mechanisms in neuronal dendrites. These results suggest that there are multiple distinct trafficking pathways and mechanisms that localize ion channels to distinct membrane sub-compartments of dendrites.
S1-5-1-3
NaチャネルとNa/Kポンプ:時間情報処理における役割
Na channel and Na/K-ATPase: roles in temporal coding in auditory neurons

○久場博司1
○Hiroshi Kuba1
名古屋大学大学院・医学系研究科・細胞生理1, JSTさきがけ2
Dept. Cell Physiol., Nagoya Univ. Grad. Sch. Med.1, JST PREST, Saitama2

In the auditory system, temporal information of sound is represented as a timing of action potentials generated at a specific phase of sound (phase-lock). Precision and reliability of this representation is extremely high, and this is achieved through several morphological and biophysical specializations of auditory neurons. One remarkable example is the synapse at nucleus magnocellularis (NM), a homologue of mammalian cochlear nucleus, relaying auditory temporal information from auditory nerve to binaural coincidence detectors for sound localization in birds. In NM, a few large end-bulb terminals are formed on the cell soma of neurons at high or middle tuning frequency regions, each releasing a large number of synaptic quanta that generate a large and rapid AMPA receptor-mediated postsynaptic potential. This synaptic potential reduces variations in the timing of each action potential to reach its threshold, and enables to preserve the timing of presynaptic action potentials effectively in postsynaptic NM neurons. However, this high-fidelity synaptic transmission would be a burden for the neurons in an economical respect, because it requires a large number of vesicles to be prepared at presynaptic terminals and a large amount of Na ions to be pumped out at the postsynaptic membrane, and therefore requires a large amount of energy particularly during high levels of activity. Our recent studies in chicken brain slices have suggested that subcellular localizations of Na channel and Na/K-ATPase at NM are strategically determined and play an important role in overcoming this disadvantage. In this symposium, I will present some data supporting this idea, and discuss how these molecules contribute to shape the signal processing of NM neurons.
S1-5-1-4
Voltage sensor function in voltage-gated channels
○Peter Larsson1
Department of Physiology and Biophysics, University of Miami Miller School of Medicine1

Members of the super family of voltage-gated cation channels all use a conserved voltage-sensing domain made up of four transmembrane segments. The voltage sensor is made up of the fourth transmembrane segment (S4), which contains a number of positively-charged amino acids. We use a fluorescence technique called voltage clamp fluorometry (VCF) to measure the movement of the voltage sensor S4 in a number of different ion channels. We have measured the movement of the voltage sensor in the cardiac KCNQ1 channels in the presence and absence of different beta subunits. The different beta subunits alter the S4 movement and the response of the channel gate to the movement of the voltage sensor. We have also studied the movement of S4 in the voltage-gated proton (VSOP or Hv1) channel in order to understand how these channels are activated by voltage. Our results suggest that in voltage-gated proton channels the two subunits activate their voltage sensor independently, but that there is a cooperative late opening step in both subunits that opens the two permeation pathways in the two subunits simultenously.
S1-5-1-5
電位センサー蛋白機能の多様性:イオン透過からホスファターゼまで
Molecular diversities of voltage sensing: from ion permeation to enzyme

○岡村康司1,2,3, 藤原祐一郎1, 坂田宗平1, 河合喬文1, 筒井秀和1,3, 大河内善史1
○Yasushi Okamura1,2,3, Yuichiro Fujiwara1, Souhei Sakata1, Takafumi Kawai1, Hidekazu Tsutsui1,3, Yoshifumi Okochi1
大阪大院・医・統合生理1, 大阪大院・生命機能2, 理研・BSI3
Dept Physiol, Osaka Univ1, Grad Frontier Biosci, Osaka Univ, Osaka, Japan2, RIKEN, BSI, Wako, Japan3

Voltage-gated ion channels operate as basic elements of electrical signals in neurons through their cation selectivity and voltage dependent gating. These are grouped into Nav channel, Kv channel, Cav channel, HCN channel, dependent on cation selectivity and voltage polarity for activating ion permeation, but they share basic molecular organization; four repeats of six transmembrane segments consisting of the first four transmembrane segments as the voltage sensor domain and the last two transmembrane segments with inner loop corresponding to the pore domain. Recent studies of X-ray crystallography and biophysical experiments have revealed detailed molecular mechanisms of operation of ion permeation and voltage sensing of human voltage-gated potassium channel and bacterial voltage-gated sodium channel. Although voltage sensor has long been studied as element specific to voltage-gated ion channels, recent studies have shown two membrane proteins in which authentic pore domain is missing. Voltage-sensing phosphatase, VSP, contains phosphoinositide phosphatase that is activated upon membrane depolarization. Voltage-sensor only protein (VSOP) or Hv1 has the voltage-sensor domain with the C-terminal coiled-coil and operates as the dimeric voltage-gated proton channel. In VSOP/Hv1, voltage sensor has dual roles: voltage sensing and ion permeation. VSOP/Hv1 is exclusively expressed in microglia, playing role in homeostasis of intracellular pH and production of reactive oxygen species. In this talk, we will show recent findings of molecular properties and physiological roles of these proteins with the voltage sensor domain. We will also demonstrate examples of applying the voltage sensor domain to develop a molecular tool as fluorescent voltage probe.

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