Takuya Hikima

Molecular Determinants Differentiating Photocurrent Properties of Two Channelrhodopsins from Chlamydomonas

A light signal is converted into an electrical one in a single molecule named channelrhodopsin, one of the archaea-type rhodopsins in unicellular green algae. Although highly homologous, two molecules of this family, channelrhodopsin-1 (ChR1) and -2 (ChR2), are distinct in photocurrent properties such as the wavelength sensitivity, desensitization, and turning-on and -off kinetics. However, the structures regulating these properties have not been completely identified. Photocurrents were analyzed for several chimera molecules made by replacing N-terminal segments of ChR2 with the homologous counterparts of ChR1. We found that the wavelength sensitivity of the photocurrent was red-shifted with negligible desensitization and slowed turning-on and -off kinetics when replacement was made with the segment containing the fifth transmembrane helix of ChR1. Therefore, this segment is involved in the determination of photocurrent properties, the wavelength sensitivity, and the kinetics characterizing ChR1 and ChR2. Eight amino acid residues differentiating this segment were exchanged one-by-one, and the photocurrent properties of each targeted mutant ChR2 were further analyzed. Among them, position Tyr226(ChR1)/Asn187(ChR2) is one of the molecular determinants involved in the wavelength sensitivity, desensitization, and turning-on and -off kinetics. It is suggested that these amino acid residues directly or indirectly interact with the chromophore as well as with the protein structure determining the photocurrent kinetics. Some of the chimera channelrhodopsins are suggested to have several advantages over the wild-type ChR2 in the introduction of light-induced membrane depolarization for the purpose of artificial stimulation of neurons in vivo and visual prosthesis for photoreceptor degeneration.

β-Phorbol ester-induced enhancement of exocytosis in large mossy fiber boutons of mouse hippocampus

Abstractβ-Phorbol esters (BPE), synthetic analogues of diacylglycerol (DAG), induce the potentiation of transmission in many kinds of synapses through activating the C1 domain-containing receptors. However, their effects on synaptic vesicle exocytosis have not yet been investigated. Here, we evaluated the vesicular exocytosis directly from individual large mossy fiber boutons (LMFBs) in hippocampal slices from transgenic mice that selectively express synaptopHluorin (SpH). We found that the activity-dependent increment of SpH fluorescence (ΔSpH) was enhanced by 4β-phorbol 12,13-diacetate (PDAc), one of the BPEs, without influencing the recycled component of SpH. These PDAc effects on ΔSpH were almost completely inhibited by staurosporine, a non-selective antagonist of protein kinases. However, intermittent synaptic transmission was still potentiated through a staurosporine-resistant mechanism. The staurosporine-sensitive cascade may facilitate the vesicle replenishment, thus maintaining the fidelity of transmission at a high level during repetitive firing of the presynaptic neuron.

Glu-97 of channelrhodopsin-2 is one of the molecular determinants involved in the ion flux

P2-p10 Glu-97 of channelrhodopsin-2 is one of the molecular determinants involved in the ion flux Toru Ishizuka1, Yuka Sugiyama2,3, Hongxia Wang1,3, Takuya Hikima1,3, Minami Sato4, Jun Kuroda4, Tetsuo Takahashi4, Hiromu Yawo1,2,3 1 Dept of Dev Biol & Neurosci, Tohoku Univ Grad Sch of Life Sci, Sendai, Japan; 2 Dept of Physiol & Pharmacol, Tohoku Univ Grad Sch of Med, Sendai, Japan; 3 Basic & Transl Res Ctr for Global Brain Sci, Tohoku Univ, Sendai, Japan; 4 Fac of Pharmaceutic Sci, Toho Univ, Funabashi, Japan

Molecular determinant differenciating Chlamydomonas channelrhodopsins

P2-p10 Glu-97 of channelrhodopsin-2 is one of the molecular determinants involved in the ion flux Toru Ishizuka1, Yuka Sugiyama2,3, Hongxia Wang1,3, Takuya Hikima1,3, Minami Sato4, Jun Kuroda4, Tetsuo Takahashi4, Hiromu Yawo1,2,3 1 Dept of Dev Biol & Neurosci, Tohoku Univ Grad Sch of Life Sci, Sendai, Japan; 2 Dept of Physiol & Pharmacol, Tohoku Univ Grad Sch of Med, Sendai, Japan; 3 Basic & Transl Res Ctr for Global Brain Sci, Tohoku Univ, Sendai, Japan; 4 Fac of Pharmaceutic Sci, Toho Univ, Funabashi, Japan

Activation of presynaptically silent synapses—One of underlying mechanisms enhancing mossy fiber transmission in the hippocampus

The activation of presynaptically silent synapses is hypothesized to be one of mechanisms of the synaptic enhancement. This hypothesis was tested for the PKC-dependent enhancement of mossy fiber (MF)-CA3 synaptic transmission in the hippocampus. Individual large MF boutons were identified in the acute slice of hippocampus and the activity-dependent changes of synaptopHluorin were measured. We found that the individual MF boutons are variably responsive to PKC. Among them some presynaptically silent boutons became active. The individual MF bouton consists of dozens of active zones. It is possible that each active zone has at least two states, releasable and non-releasable. These MF boutons may have mostly non-releasable active zones which become releasable by a PKC-dependent mechanism. All animal procedures were conducted in accordance with the guiding principles of NIH.

Vesicular dynamics and its plasticity in the hippocampal synapse

Hippocampal mossy fibers, axons of dentate granule cells, run through the dentate hilus toward the CA3 region and make synapses with hilar and CA3 neurons. In the hippocampus of temporal lobe epilepsy patients and its animal models, mossy fibers intensely branch in the hilus and project oppositely to the molecular layer, where they provide a large number of excitatory synaptic contacts with the dendrites of granule cells. This abnormal development of mossy fibers, termed the mossy fiber sprouting, likely exacerbate the disease by forming hyperexcitable recurrent loops in the dentate gyrus that cause epileptiform burst firings of granule cells. My colleagues and I revealed that brain-derived neurotrophic factor (BDNF) induces branching of mossy fibers and are now investigating the role of cyclic adenosine monophosphate (cAMP) in regulating the direction of mossy fiber projections. In this talk, I will review the axon guidance mechanism of mossy fibers and discuss how they are misguided to the opposite direction under epileptic conditions, referring to our recent findings.