DEPARTMENT OF CELL PHYSIOLOGY

Outline

Department of Cell Physiology is composed of four divisions, Division of Membrane Physiology, Correlative Physiology, Neural Systematics, and Cell Signaling. Divisions of Neural Systematics and Cell Signaling are adjunct and joint divisions, respectively. To clarify roles of channels, transporters, receptors, biosensor channels (TRPs), enzymes and their protein complexes in physiological functions, researches are being done mainly at the levels ranging from the molecule, cell, and tissue to organ. In addition, researches are being conducted to elucidate the pathological mechanisms associated with their dysfunction. Furthermore, studies on regulatory mechanisms for processing visual information in retina and sleep/wakefulness are being done.

Each division has the latest research technology and methods on molecular and cellular biology, biochemistry and proteomics, electrophysiology, neuroanatomy, and molecular genetics. Sharing and developing these technology and methods, we are attempting to reveal the physiological function of human body in an integrative manner.

Research focuses on membrane proteins and especially on the functional proteins localized in the synaptic membranes of the brain (neurotransmitter receptors, ion channels, adhesion molecules, and so on). Synaptic membrane proteins do not function alone, but form protein complexes (functional units) with anchoring proteins, signaling proteins, and thereby performing their physiological functions. Many synaptic proteins undergo protein palmitoylation, and the localization on the synaptic membrane is dynamically regulated by neuronal activity. The goal is to elucidate the molecular mechanism determining the dynamic localization for synaptic proteins, by the functional analyses of the palmitoylating enzymes we recently discovered. In addition, taking advantage of our unique biochemical techniques, we are purifying and identifying novel synaptic protein complexes from the brain tissue, in particular focusing on disease candidate proteins. Based on these molecular clues, we will elucidate the molecular mechanism for controlling synaptic transmission by multidisciplinary approaches.

Figure 1. Palmitoylation determines the localization of various synaptic proteins.
Protein palmitoylation is a common posttranslational lipid modification and regulates the membrane targeting of proteins such as a postsynaptic scaffold, PSD-95. Palmitoylation is unique in that it is reversible and dynamically regulated by specific extracellular signals. The reversible nature of protein palmitoylation allows proteins to shuttle between intracellular compartments. (A) For example, wild-type PSD-95 clearly targets to postsynaptic membrane in hippocampal neurons (left), whereas palmitoylation deficient mutant of PSD-95 diffusely localizes in somato-dendrites (right). (B) Upon activity blockade, a palmitoylation enzyme translocates to postsynaptic density and induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA-type glutamate receprotors. We are currently challenging the question how dynamic cycling between palmitoylation and depalmitoylation is regulated inside living neurons.

Figure 2. Synaptic proteins do not usually function alone, but form protein complexes with anchoring proteins and signaling proteins, thereby performing their physiological functions. Taking advantage of our unique biochemical approaches, we have identified various physiological protein complexes. Examples include PSD-95- and LGI1-containing protein complexes. LGI1 is a neuronal secreted protein whose mutations were reported in patients with an inherited form of human epilepsy. We recently purified the LGI1-containing protein complexes from mouse brain, and found that secreted protein LGI1 functions as a ligand for ADAM22 and ADAM23. Interestingly loss of LGI1, ADAM22, or ADAM23 in mice showed similar epileptic seizures. Also, loss of LGI1 reduces AMPA receptor-mediated synaptic transmission.
Thus, we will clarify the major protein-protein networks and the various physiological functions, such as synaptic transmission in the brain.

Staff

All of the cell functions, including neural activities, are performed or supported by operation of bio-molecular sensors, channels (ion and water channels) and transporters (carriers and pumps) located on the membrane. The objectives of our division work are to elucidate molecular mechanisms of most general cell activities, such as volume regulation, absorption/secretion and environmental signal reception, to clarify roles of channels, transporters and receptors in these fundamental functions from the viewpoint of integrative biology, and to throw the light on the relationship between these malfunctions and diseases or cell death, as well as to study the multifunctionality of channel and transporter during cell functions or malfunctions.

The main subjects of our current research are as follows:

(1) "Mechanisms of processing visual information in retina" In the retina, various kinds of bio-molecular sensors express in retinal neurons to form early vision. We investigate visual function in retina, especially visual integration mechanisms in retinal ganglion cells by gene-manipulation of bio-molecular sensors and electrophysiology with organotypic retinal culture system (Fig. 1).

(2) "Molecular mechanisms of cell volume regulation and their physiological roles": Most cells regulate their cell volume even under anisotonic conditions. In the volume regulation mechanisms, a number of channels, transporters and receptors are involved (Fig. 2). We are investigating to identify volume-regulatory membrane machineries, including the volume-sensitive anion channel, and to clarify their physiological roles.

(3) "Induction mechanisms of apoptotic, necrotic and ischemic cell death": Dysfunction of cell volume regulation is associated with necrotic and apoptotic cell death (Fig. 3) which is coupled to persistent swelling (necrotic volume increase: NVI) and shrinkage (apoptotic volume decrease: AVD). Our aim is to pioneer the new field of 'PHYSIOLOGY OF CELL DEATH' through elucidation of the mechanisms of cell volume regulation and their dysfunction. We are attempting to focus our studies on the mechanisms of ischemic cell death of brain neurons and cardiac myocytes.

(4) "Molecular mechanisms of channel functions as bio-molecular sensors": Channels are multifunctional proteins involved not only in electric signal generation and ion transport but also in sensing the environmental factors or stress. We aim at elucidating molecular mechanisms of volume- and stress-sensing functions of anion channels, ATP channels and TRP cation channels (Fig. 4).

Fig. 1 Oraganotypic culture of adult retina and gene transfection [after Koizumi et al, 2007, PLoS One]

Fig. 2 Molecular mechanisms of the regulatory volume decrease (RVD) and of volume-sensor Cl channel (VSOR) activation. [after Okada et al. 2001, J. Physiol. 532, 3-16]

Fig. 3 Roles of channels and transporters in the induction of apoptotic volume decrease (AVD) and apoptotic cell death as well as in that of necrotic volume increase (NVI) and necrotic cell death. [after Okada et al. 2009, J Physiol.]

Fig. 4 Stress-sensing, ATP-releasing maxi-anion channel in cardiomyocytes. [after Sabirov & Okada 2005, Purinergic Signalling. 1, 311-328]

Staff

Division of Neural Systematics

Division of Cell Signaling

Department of Bio-Environmental Science,
OKAZAKI INSTITUTE FOR
INTEGRATIVE BIOSCIENCE

We mainly investigate molecular mechanisms of thermosensation, nociception, taste sensation and Ca²⁺ absorption by focusing on TRP ion channels. We also investigate molecular mechanisms of sleep/wakefulness regulation by focusing on orexin neurons. Molecular cell biological, biochemical, developmental biological and electrophysiological techniques are utilized to achieve the above objectives. The followings are major projects in progress.

(1) Molecular mechanisms of thermosensation: Temperature sensing ability is conferred by ion channels of the TRPV, TRPM and TRPA families. We try to clarify the molecular mechanisms of thermosensation and their physiological significance by focusing on those thermosensitive TRP channels from mammals to insects. We are also doing behavioral analyses of mice lacking TRPV3, TRPV4 or TRPM2. Furthermore, we are trying to isolate a novel thermosensitive TRP channels.

(2) Molecular mechanisms of nociception: Capsaicin receptor TRPV1 and TRPA1 are ion channels activated by different noxious stimuli. We try to clarify the nociceptive mechanisms at peripheral nerve endings by focusing on TRP ion channels, especially TRPV1 and TRPA1. We are also doing behavioral analyses of TRPV1- or TRPA1-deficient mice.

(3) We study neurons in the hypothalamus. The hypothalamus is implicated in the maintenance of homeostasis, such as body temperature regulation, feeding regulation and sleep/wakefulness regulation. We make transgenic animals and analyze them by using many techniques including electrophysiological analysis such as slice patch clamp and in vivo extracellular recording as well as immunohistochemical analysis and behavioral analysis such as sleep recording. We try to reveal the neural mechanism, which involved in the maintenance of homeostasis, in molecular, cellular and whole animal level.

[Mammalian thermosensitive TRP channels (upper) and their properties (lower)]

[Studying the regulatory mechanisms involved in feeding and sleep/wakefulness at multi levels: molecular, cellular and whole animal levels]

Staff