Researchers Database

nakajou kouichi

    PhysiologyIntegrativePhysiology Professor
Last Updated :2021/10/17

Researcher Information

Degree

  • PhD(Univ of Tokyo)

URL

Research funding number

  • 80390699

ORCID ID

J-Global ID

Research Interests

  • zebrafish   voltage-gated ion channel   electrophysiology   single molecule imaging   voltage sensor   potassium channels   ion channels   

Research Areas

  • Life sciences / Physiology
  • Life sciences / Neuroscience - general
  • Life sciences / Biophysics

Academic & Professional Experience

  • 2018/04 - Today  Jichi Medical UniversityDivision of Integrative PhysiologyProfessor
  • 2015/03 - 2018/03  Osaka Medical CollegeDepartment of PhysiologyAssociate Professor
  • 2005/11 - 2015/02  SOKENDAIDepartment of Physiological SciencesAssistant Professor
  • 2005/04 - 2015/02  National Institute for Physiological SciencesDivision of Biophysics and NeurobiologyAssistant Professor
  • 2008/09 - 2009/03  UC BerkeleyVisiting researcher
  • 2004/04 - 2005/03  National Institute for Physiological SciencesDivision of Biophysics and NeurobiologyPostdoc
  • 2002/04 - 2004/03  Tokyo Medical and Dental UnivPostdoc
  • 1999/04 - 2002/03  学術振興会特別研究員(DC1)
  • 1999/06 - 1999/09  Case Western Reserve Univ.Visiting scholar

Education

  • 1997/04 - 2002/03  The University of Tokyo  総合文化研究科  広域科学専攻
  • 1994/04 - 1997/03  The University of Tokyo  College of Arts and Sciences  Department of Basic Science
  • 1992/04 - 1994/03  The University of Tokyo  College of Arts and Sciences  理科二類

Association Memberships

  • PHYSIOLOGICAL SOCIETY OF JAPAN   THE BIOPHYSICAL SOCIETY OF JAPAN   Biophysical Society   日本神経科学学会   

Published Papers

  • Yoshiaki Kise, Go Kasuya, Hiroyuki H Okamoto, Daichi Yamanouchi, Kan Kobayashi, Tsukasa Kusakizako, Tomohiro Nishizawa, Koichi Nakajo, Osamu Nureki
    Nature 2021/09 [Refereed]
     
    Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits-intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)-to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1-5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2-DPP6S-KChIP1 dodecamer complex, the Kv4.2-KChIP1 and Kv4.2-DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2-KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1-S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2-KChIP1-DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.
  • Fujii K, Nakajo K, Egashira Y, Yamamoto Y, Kitada K, Taniguchi K, Kawai M, Tomiyama H, Kawakami K, Uchiyama K, Ono F
    Cell Reports 30 (9) 2879 - 2888 2211-1247 2020/03 [Refereed][Not invited]
  • Nakajo K
    Biophysics and Physicobiology 16 121 - 126 2189-4779 2019/05 [Refereed][Invited]
  • Koichi NAKAJO
    Seibutsu Butsuri 58 (3) 144 - 148 2018/06 [Refereed][Invited]
  • Hisao Tsukamoto, Masahiro Higashi, Hideyoshi Motoki, Hiroki Watanabe, Christian Ganser, Koichi Nakajo, Yoshihiro Kubo, Takayuki Uchihashi, Yuji Furutani
    Journal of Biological Chemistry 293 (18) 6969 - 6984 1083-351X 2018 [Refereed][Not invited]
     
    Canonical K channels are tetrameric and highly K-selective, whereas two-pore– domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore– domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange–induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm1. This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm1 band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1’s surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful “molecular fingerprints” for assessing the properties of other K channels.
  • Akiyuki Taruno, Hongxin Sun, Koichi Nakajo, Tatsuro Murakami, Yasuyoshi Ohsaki, Mizuho A. Kido, Fumihito Ono, Yoshinori Marunaka
    JOURNAL OF PHYSIOLOGY-LONDON 595 (18) 6121 - 6145 0022-3751 2017/09 [Refereed][Not invited]
     
    Key points Calcium homeostasis modulator 1 (CALHM1), a new voltage-gated ATP- and Ca2+-permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post-translationally regulated through the process of protein S-palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine-tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory. Emerging roles of CALHM1, a recently discovered voltage-gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post-translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S-palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance-voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent-resistant membranes. Our results comprehensively uncover a post-translational regulation of the voltage-dependent gating of CALHM1 by palmitoylation.
  • Masahiro Kitazawa, Yoshihiro Kubo, Koichi Nakajo
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 (37) 22724 - 22733 0021-9258 2015/09 [Refereed][Not invited]
     
    Kv4 is a member of the voltage-gated K+ channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4: 2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.
  • Koichi Nakajo, Yoshihiro Kubo
    JOURNAL OF PHYSIOLOGY-LONDON 593 (12) 2617 - 2625 0022-3751 2015/06 [Refereed][Invited]
     
    The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel.
  • Koichi Nakajo, Yoshihiro Kubo
    NATURE COMMUNICATIONS 5 2041-1723 2014/06 [Refereed][Not invited]
     
    In voltage-gated K+ channels, membrane depolarization induces an upward movement of the voltage-sensing domains (VSD) that triggers pore opening. KCNQ1 is a voltage-gated K+ channel and its gating behaviour is substantially modulated by auxiliary subunit KCNE proteins. KCNE1, for example, markedly shifts the voltage dependence of KCNQ1 towards the positive direction and slows down the activation kinetics. Here we identify two phenylalanine residues on KCNQ1, Phe232 on S4 (VSD) and Phe279 on S5 (pore domain) to be responsible for the gating modulation by KCNE1. Phe232 collides with Phe279 during the course of the VSD movement and hinders KCNQ1 channel from opening in the presence of KCNE1. This steric hindrance caused by the bulky amino-acid residues destabilizes the open state and thus shifts the voltage dependence of KCNQ1/KCNE1 channel.
  • Masahiro Kitazawa, Yoshihiro Kubo, Koichi Nakajo
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 (25) 17597 - 17609 0021-9258 2014/06 [Refereed][Not invited]
     
    Kv4 is a voltage-gated K+ channel, which underlies somatodendritic subthreshold A-type current (I-SA) and cardiac transient outward K+ (I-to) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K+ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4.KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2.KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4.KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.
  • Koichi NAKAJO, Yoshihiro KUBO
    Seibutsu Butsuri 53 (6) 313 - 6 2013/01 [Refereed][Invited]
  • Koichi Nakajo, Atsuo Nishino, Yasushi Okamura, Yoshihiro Kubo
    JOURNAL OF GENERAL PHYSIOLOGY 138 (5) 521 - 535 0022-1295 2011/11 [Refereed][Not invited]
     
    KCNQ1 channels are voltage-gated potassium channels that are widely expressed in various non-neuronal tissues, such as the heart, pancreas, and intestine. KCNE proteins are known as the auxiliary subunits for KCNQ1 channels. The effects and functions of the different KCNE proteins on KCNQ1 modulation are various; the KCNQ1-KCNE1 ion channel complex produces a slowly activating potassium channel that is crucial for heartbeat regulation, while the KCNE3 protein makes KCNQ1 channels constitutively active, which is important for K+ and Cl-. transport in the intestine. The mechanisms by which KCNE proteins modulate KCNQ1 channels have long been studied and discussed; however, it is not well understood how different KCNE proteins exert considerably different effects on KCNQ1 channels. Here, we approached this point by taking advantage of the recently isolated Ci-KCNQ1, a KCNQ1 homologue from marine invertebrate Ciona intestinalis. We found that Ci-KCNQ1 alone could be expressed in Xenopus laevis oocytes and produced a voltage-dependent potassium current, but that Ci-KCNQ1 was not properly modulated by KCNE1 and totally unaffected by coexpression of KCNE3. By making chimeras of Ci-KCNQ1 and human KCNQ1, we determined several amino acid residues located in the pore region of human KCNQ1 involved in KCNE1 modulation. Interestingly, though, these amino acid residues of the pore region are not important for KCNE3 modulation, and we subsequently found that the S1 segment plays an important role in making KCNQ1 channels constitutively active by KCNE3. Our findings indicate that different KCNE proteins use different domains of KCNQ1 channels, and that may explain why different KCNE proteins give quite different outcomes by forming a complex with KCNQ1 channels.
  • Koichi Nakajo, Yoshihiro Kubo
    CHANNELS 5 (5) 395 - 399 1933-6950 2011/09 [Refereed][Invited]
     
    The KCNQ1 channel is a voltage-dependent potassium channel, which is widely expressed in various tissues of the human body including heart, inner ear, intestine, kidney and pancreas. The ion channel properties of KCNQ1 change remarkably when auxiliary subunit KCNE proteins co-exist. The mechanisms of KCNQ1 channel regulation by KCNE proteins are of longstanding interest but are still far from being fully understood. The pore region (S5-S6 segments) of KCNQ1 is thought to be the main interaction site for KCNE proteins. However, some recent reports showed that the voltage-sensing domain (S1-S4 segments) is critically involved in the regulation of KCNQ1 by KCNE proteins. In addition, we recently re-examined the stoichiometry of the KCNQ1-KCNE1 complex and found that the stoichiometry is not fixed but rather flexible and the KCNQ1 channel can have up to four associated KCNE1 proteins. We will review these recent findings concerning the mechanisms of KCNQ1 regulation by KCNE proteins.
  • Katsuhiro Nagatomo, Hiroshi Ishii, Tomomi Yamamoto, Koichi Nakajo, Yoshihiro Kubo
    BIOPHYSICAL JOURNAL 99 (11) 3609 - 3618 0006-3495 2010/12 [Refereed][Not invited]
     
    The transient receptor potential A1 channel (TRPA1) is activated by various compounds including isothiocya nates menthol and cinnamaldehyde The sensitivities of the rodent and human isoforms of TRPA1 to menthol and the cysteine attacking compound CMP1 differ and the molecular determinants for these differences have been identified in the 5th transmembrane region (TM5) for menthol and TM6 for CMP1 We recently reported that caffeine activates mouse TRPA1 (mTRPA1) but suppresses human TRPA1 (hTRPA1) Here we aimed to identify the molecular determinant that is responsible for species specific differences in the response to caffeine by analyzing the functional properties of various chimeras expressed in Xenopus oocytes We initially found that the region between amino acids 231 and 287 in the distal N terminal cytoplasmic region of mTRPA1 is critical In a mutagenesis study of this region we subsequently observed that introduction of a Met268Pro point mutation into mTRPA1 changed the effect of caffeine from activation to suppression Because the region including Met 268 is different from other reported ligand binding sites and from the EF hand motif these results suggest that the caffeine response is mediated by a unique mechanism and confirm the importance of the distal N terminal region for regulation of TRPA1 channel activity
  • Yoshihiro Kubo, Yuichiro Fujiwara, Batu Keceli, Koichi Nakajo
    Brain and Nerve 62 (12) 1323 - 1329 1881-6096 2010/12 [Refereed][Not invited]
     
    ATP is known to function as a neurotransmitter. There are 2 major families of ATP receptors-the ion channel-type P2X receptors and metabotropic P2Y receptors. P2X receptors are known to possess unique properties of pore dilation that depends on the time lapse after ATP application further, they exert their functions by directly interacting with nicotinic ACh receptors. These properties suggest the flexibility of the pore formed by these receptors. We studied the biophysical properties of P2X2 receptor by using in vitro expression systems and focused on various dynamic regulations and structural rearrangements. Firstly, the pore property clearly depended on the expression levels of the P2X2 receptors on the membrane. When the expression level was high, inward rectification was weak, and pore dilation was clearly observed. We also clarified that the key feature of the pore property is not the number of channels expressed but the number of open channels on the membrane. Secondly, we focused on the regulation of these channels by phosphoinositides (PIPns). PIPns are known to regulate the activity of various ion channels, but in the case of P2X2, we observed that PIPns regulate not only the activity but also the processes of pore dilation and desensitization. The binding of PIPns to P2X2 in the pore dilated state was observed to be less stable. Application of a reagent, which decreases the levels of PIPns, and mutation of the binding site facilitated desensitization of P2X2 in the pore dilated state. Thirdly, we analyzed the voltage-dependent gating of these channels. Although P2X2 lacks a canonical voltage-sensor domain, it undergoes voltage-dependent activation upon hyperpolarization. Further, we observed that the voltage-dependent gating depends on ATP concentration conductance-voltage relationship curve shifted toward depolarization potential with increase in ATP concentration. We found that the ATP-binding site and the extracellular sideus of the transmembrane region were critical for the voltage-dependent gating. These results show that P2X2 channel pore is exceptionally flexible, and that the channel activity is dynamically regulated by various factors, including not only ATP but also PIPns and membrane potential.
  • Koichi Nakajo, Maximilian H. Ulbrich, Yoshihiro Kubo, Ehud Y. Isacoff
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 (44) 18862 - 18867 0027-8424 2010/11 [Refereed][Not invited]
     
    The KCNQ1 voltage-gated potassium channel and its auxiliary subunit KCNE1 play a crucial role in the regulation of the heartbeat. The stoichiometry of KCNQ1 and KCNE1 complex has been debated, with some results suggesting that the four KCNQ1 subunits that form the channel associate with two KCNE1 subunits (a 4: 2 stoichiometry), while others have suggested that the stoichiometry may not be fixed. We applied a single molecule fluorescence bleaching method to count subunits in many individual complexes and found that the stoichiometry of the KCNQ1 - KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (a 4: 4 stoichiometry). The proportion of the various stoichiometries was found to depend on the relative expression densities of KCNQ1 and KCNE1. Strikingly, both the voltage-dependence and kinetics of gating were found to depend on the relative densities of KCNQ1 and KCNE1, suggesting the heart rhythm may be regulated by the relative expression of the auxiliary subunit and the resulting stoichiometry of the channel complex.
  • Hiroshi Ishii, Koichi Nakajo, Yuchio Yanagawa, Yoshihiro Kubo
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (5) 736 - 748 0953-816X 2010/09 [Refereed][Not invited]
     
    The mouse cerebellum consists of 10 lobules, which are distinguishable by their anatomical and functional properties. However, the differences in the slow postsynaptic currents (sPSCs) of Purkinje cells between lobules have not been well studied. We recorded the sPSCs of lobules 3, 9 and 10 evoked by tetanic stimulation of the molecular layer in cerebellar slices, and found a novel outward sPSC mediated by the GABA(B) receptor in loblues 9 and 10 but hardly at all in lobule 3. We showed that the lobule-specific difference is at least partly attributable to differences in the density of GABAergic neurons (higher in lobule 10 than in lobules 3 and 9), and the functional expression level of postsynaptic GABA(B) receptor currents (larger in lobules 9 and 10 than in lobule 3). The G-protein-coupled inward rectifying K+ channel (GIRK) is known to be activated by GABA(B) receptors; however, the outward sPSC was not blocked by a GIRK blocker, was not sensitive to Cs+ block, and was observed when Cs+ was used as a charge carrier. These results suggest that a K+ channel other than GIRK could be activated by GABA(B) receptors. KCNK13 is a Cs+-permeable K+ channel that shows intense expression of mRNA in Purkinje cells. KCNK13 current was enhanced by co-expression of G(beta gamma) subunits and was observed when Cs+ was used as a charge carrier in heterologous expression systems, and the amino acids critical for these features were identified by mutagenesis. Taken together, these results show that KCNK13 is a legitimate candidate for the Cs+-permeable K+ channel activated by GABA(B) receptors, presumably via G(beta gamma) subunits in Purkinje cells.
  • Yoshihiro Kubo, Yuichiro Fujiwara, Batu Keceli, Koichi Nakajo
    JOURNAL OF PHYSIOLOGY-LONDON 587 (22) 5317 - 5324 0022-3751 2009/11 [Refereed][Not invited]
     
    The P2X(2) channel is a ligand-gated channel activated by ATP. Functional features that reflect the dynamic flexibility of the channel include time-dependent pore dilatation following ATP application and direct inhibitory interaction with activated nicotinic acetylcholine receptors on the membrane. We have been studying the mechanisms by which P2X(2) channel functionality is dynamically regulated. Using a Xenopus oocyte expression system, we observed that the pore properties, including ion selectivity and rectification, depend on the open channel density on the membrane. Pore dilatation was apparent when the open channel density was high and inward rectification was modest. We also observed that P2X(2) channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X(2) channels were activated upon membrane hyperpolarization. This voltage-dependent activation was also [ATP] dependent. With increases in [ATP], the speed of hyperpolarization-induced activation was increased and the conductance-voltage relationship was shifted towards depolarized potentials. Based on analyses of experimental data and various simulations, we propose that these phenomena can be explained by assuming a fast ATP binding step and a rate-limiting voltage-dependent gating step. Complete elucidation of these regulatory mechanisms awaits dynamic imaging of functioning P2X(2) channels.
  • Yuichiro Fujiwara, Batu Keceli, Koichi Nakajo, Yoshihiro Kubo
    JOURNAL OF GENERAL PHYSIOLOGY 133 (1) 93 - 109 0022-1295 2009/01 [Refereed][Not invited]
     
    P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance-voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X(2)-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].
  • Alexis S. Hill, Atsuo Nishino, Koichi Nakajo, Giuxin Zhang, Jaime R. Fineman, Michael E. Selzer, Yasushi Okamura, Edward C. Cooper
    PLOS GENETICS 4 (12) 1553-7390 2008/12 [Refereed][Not invited]
     
    In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin-spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features ( lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.
  • Koichi Nakajo, Yoshihiro Kubo
    JOURNAL OF PHYSIOLOGY-LONDON 586 (12) 2827 - 2840 0022-3751 2008/06 [Refereed][Not invited]
     
    KCNQ channels carry the slowly activating, voltage-dependent M-current in excitable cells such as neurons. Although the KCNQ2 homomultimer can form a functional voltage-gated K+ channel, heteromultimerization with KCNQ3 produces a > 10-fold increase in current amplitude. All KCNQ channels contain double coiled-coil domains (TCC1 and TCC2, or A-domain Head and Tail), of which TCC2 (A-domain Tail) is thought to be important for subunit recognition, channel assembly and surface expression. The mechanism by which TCC2 recognizes and associates with its partner is not fully understood, however. Our aim in the present study was to elucidate the recognition mechanism by examining the phenotypes of TCC2-deletion mutants, TCC2-swapped chimeras and point mutants. Electrophysiological analysis using Xenopus oocytes under two-electrode voltage clamp revealed that homotetrameric KCNQ3 TCC2 is a negative regulator of current expression in the absence of KCNQ2 TCC2. Recent structural analysis of KCNQ4 TCC2 revealed the presence of intercoil salt bridge networks. We therefore swapped the sign of the charged residues reportedly involved in the salt bridge formation and functionally confirmed that the intercoil salt bridge network is responsible for the subunit recognition between KCNQ2 and KCNQ3. Finally, we constructed TCC2-swapped KCNQ2/KCNQ3 mutants with KCNQ1 TCC2 or GCN4-pLI, a coiled-coil domain from an unrelated protein, and found that TCC2 is substitutable and even GCN4-pLI can work as a substitute for TCC2. Our present data provide some new insights into the role played by TCC2 during current expression, and also provide functional evidence of the importance of the intercoil salt bridge network for subunit recognition and coiled-coil formation, as is suggested by recent crystallographic data.
  • Koichi Nakajo, Yoshihiro Kubo
    JOURNAL OF GENERAL PHYSIOLOGY 130 (3) 269 - 281 0022-1295 2007/09 [Refereed][Not invited]
     
    KCNQ1 is a voltage-dependent K+ channel whose gating properties are dramatically altered by association with auxiliary KCNE proteins. For example, KCNE1, which is mainly expressed in heart and inner ear, markedly slows the activation kinetics of KCNQ1. Whether the voltage-sensing S4 segment moves differently in the presence of KCNE1 is not yet known, however. To address that question, we systematically introduced cysteine mutations, one at a time, into the first half of the S4 segment of human KCNQ1. A226C was found out as the most suited mutant for a methanethiosulfonate (MTS) accessibility analysis because it is located at the N-terminal end of S4 segment and its current was stable with repetitive stimuli in the absence of MTS reagent. MTS accessibility analysis revealed that the apparent second order rate constant for modification of the A226C mutant was state dependent, with faster modification during depolarization, and was 13 times slower in the presence of KCNE1 than in its absence. In the presence of KCNE3, on the other hand, the second order rate constant for modification was not state dependent, indicating that the C226 residue was always exposed to the extracellular milieu, even at the resting membrane potential. Taken together, these results suggest that KCNE1 stabilizes the S4 segment in the resting state and slows the rate of transition to the active state, while KCNE3 stabilizes the S4 segment in the active state. These results offer new insight into the mechanism of KCNQ1 channel modulation by KCNE1 and KCNE3.
  • K Nakajo, Y Kubo
    JOURNAL OF PHYSIOLOGY-LONDON 569 (1) 59 - 74 0022-3751 2005/11 [Refereed][Not invited]
     
    It is well established that stimulation of G(q)-coupled receptors such as the M1 muscarinic acetylcholine receptor inhibits KCNQ/M currents. While it is generally accepted that this muscarinic inhibition is mainly caused by the breakdown Of PIP2, the role of the subsequent activation of protein kinase C (PKC) is not well understood. By reconstituting M currents in Xenopus oocytes, we observed that stimulation of coexpressed M1 receptors with 10 mu m oxotremorine M (oxo-M) induces a positive shift (4-30 mV, depending on which KCNQ channels are expressed) in the conductance-voltage relationship (G-V) of KCNQ channels. When we applied phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, we observed a large positive shift (17.8 +/- 1.6 mV) in the G-V curve for KCNQ2, while chelerythrine, a PKC inhibitor, attenuated the shift caused by the stimulation of M1 receptors. By contrast, reducing PIP2 had little effect on the G-V curve for KCNQ2 channels; although pretreating cells with 10 mu m wortmannin for 30 min reduced KCNQ2 current amplitude by 80%, the G-V curve was shifted only slightly (5 mV). Apparently, the shift induced by muscarinic stimulation in Xenopus oocytes was mainly caused by PKC activation. When KCNQ2/3 channels were expressed in HEK 293T cells, the G-V curve seemed already to be shifted in a positive direction, even before activation of PKC, and PMA failed to shift the curve any further. That alkaline phosphatase in the patch pipette shifted the G-V curve in a negative direction suggests KCNQ2/3 channels are constitutively phosphorylated in HEK 293T cells.
  • Y Okamura, A Nishino, Y Murata, K Nakajo, H Iwasaki, Y Ohtsuka, M Tanaka-Kunishima, N Takahashi, Y Hara, T Yoshida, M Nishida, H Okado, H Watari, IA Meinertzhagen, N Satoh, K Takahashi, Y Satou, Y Okada, Y Mori
    PHYSIOLOGICAL GENOMICS 22 (3) 269 - 282 1094-8341 2005/08 [Refereed][Not invited]
     
    Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so- called " prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/ vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate- unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/ genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome- wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.
  • K Nakajo, Y Okamura
    JOURNAL OF NEUROPHYSIOLOGY 92 (2) 1056 - 1066 0022-3077 2004/08 [Refereed][Not invited]
     
    Isolated ascidian Halocynthia roretzi blastomeres of the muscle lineage exhibit muscle cell-like excitability on differentiation despite the arrest of cell cleavage early in development. This characteristic provides a unique opportunity to track changes in ion channel expression during muscle cell differentiation. Here, we show that the intrinsic membrane property of ascidian cleavage-arrested muscle-type cells becomes oscillatory by expressing transient outward currents (I-to) activated by Ca2+- induced Ca2+ release (CICR) in a maturation-dependent manner. In current-clamp mode, most day 4 (72 h after fertilization) cleavage-arrested muscle cells exhibited an oscillatory membrane potential of -20 mV at 15 Hz, whereas most day 3 (48 h after fertilization) cells exhibited a spiking pattern. In voltage-clamp mode, the day 4 cells exhibited prominent transient outward currents that were not present in day 3 cells. I-to was abolished by the application of 10 mM caffeine, implying that CICR was involved in I-to activation. I-to was based on K+ efflux and sensitive to tetraethylammonium and some Ca2+-activated K+ channel inhibitors. We found a 60-pS single channel conductance that was activated by local Ca2+ release in ascidian muscle cell. Voltage-clamp recording with an oscillatory waveform as a command pulse showed that CICR-activated K+ currents were activated during the falling phase of the membrane potential oscillation. These results suggest that developmental expression of CICR-activated K+ current plays a role in the maturation of larval locomotion by modifying the intrinsic membrane excitability of muscle cells.
  • Y Okamura, H Izumi-Nakaseko, K Nakajo, Y Ohtsuka, T Ebihara
    NEUROSIGNALS 12 (3) 142 - 158 1424-862X 2003/05 [Refereed][Not invited]
     
    This review describes recent findings on voltage-gated Ca channel (Cav channel) cloned from ascidians, the most primitive chordates. Ascidian L-type like Cav channel has several unusual features: (1) it is closely related to the prototype of chordate L-type Cav channels by sequence alignment; (2) it is resistant to dihydropyridine due to single amino acid change in the pore region, and (3) maternally provided RNA putatively encodes a truncated protein which has remarkable suppressive effect on Cav channel expression during development. Ascidian Cav channel will provide a useful molecular clue in the future to understand Ca(2+)-regulated cell differentiation and physiology with the background of recently defined ascidian genome and molecular biological tools. Copyright (C) 2003 S. Karger AG, Basel.
  • K Nakajo, Y Katsuyama, F Ono, Y Ohtsuka, Y Okamura
    NEUROSCIENCE RESEARCH 45 (1) 59 - 70 0168-0102 2003/01 [Refereed][Not invited]
     
    Ascidians belong to the primitive chordates and their larvae show symmetrical beating of the tail, which is reminiscent of the swimming pattern in primitive vertebrates. Since ascidian larva contains only a small number of neurons in their entire larval nervous system, they will potentially provide a simple model for the study of animal locomotion. In a step towards the goal of establishing the molecular basis underlying ascidian larval neurophysiology, we describe here a Kv4 class of voltage-gated potassium channel, TuKv4, from Halocynthia roretzi. Whole mount in situ hybridization indicates that TuKv4 is expressed in most of larval neurons including motor neurons. TuKv4-currents reconstituted in Xenopus oocytes show currents with similar properties to the lower-threshold A-type currents from cleavage-arrested ascidian blastomeres of neural lineage. However, the voltage-dependency of the steady-state inactivation and activation was shifted leftward by 20 mV, as compared with native A-type currents, suggesting that other components may be required to restore full function of the Kv4 channel. Unexpectedly, another isoform lacking C-terminal cytoplasmic region was also isolated. This truncated isoform did not lead to a functional current in Xenopus oocytes. RT-PCR analysis showed that the truncated form is transiently expressed during larval development, suggesting some developmental role for potassium channel expression. (C) 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved.
  • R Okagaki, H Izumi, T Okada, H Nagahora, K Nakajo, Y Okamura
    DEVELOPMENTAL BIOLOGY 230 (2) 258 - 277 0012-1606 2001/02 [Refereed][Not invited]
     
    Ca2+ entry during electrical activity plays several critical roles in development. However, the mechanisms that regulate Ca2+ influx-during early embryogenesis remain unknown. In ascidians, a primitive chordate, development is rapid and blastomeres of the muscle and neuronal lineages are easily identified, providing a simple model for studying the expression of voltage-dependent Ca2+ channels (VDCCs) in cell differentiation. Here we isolate an ascidian cDNA, TuCa1, a homologue of the alpha (1)-subunit of L-type class Ca2+ channels. We unexpectedly found another form of Ca2+ channel cDNA (3-domain-type) potentially encoding a truncated type which lacked the first domain and a part of the second domain. An analysis of genomic sequence suggested that 3-domain-type RNA and the full-length type have alternative transcriptional start sites. The temporal pattern of the amount of 3-domain-type RNA was the reverse of that of the full-length type; the 3-domain type was provided maternally and persisted during early embryogenesis, whereas the full-length type was expressed zygotically in neuronal and muscular lineage cells. Switching of the two forms occurred at a critical stage when VDCC currents appeared in neuronal or muscular blastomeres. To examine the functional roles of the 3-domain type, it was coexpressed with the full-length type in Xenopus oocyte. The 3-domain type did not produce a functional VDCC current, whereas it had a remarkable inhibitory effect on the functional expression of the full-length form. In addition, overexpression of the 3-domain type under the control of the muscle-specific actin promoter in ascidian muscle blastomeres led to a significant decrease in endogenous VDCC currents. These findings raise the possibility that the 3-domain type has some regulatory role in tuning current amplitudes of VDCCs during early development. (C) 2001 Academic Press.
  • F Ono, Y Katsuyama, K Nakajo, Y Okamura
    JOURNAL OF NEUROSCIENCE 19 (16) 6874 - 6886 0270-6474 1999/08 [Refereed][Not invited]
     
    Na+ and K+ channels are the two key proteins that shape the action potentials in neurons. However, little is known about how the expression of these two channels is coordinated. To address this issue, we cloned a Shab-related K+ channel gene from ascidian Halocynthia roretzi (TuKv2). In this animal, a blastomere of neuronal lineage isolated from the 8-cell embryo expresses single Na+ channel and K+ channel genes after neural induction. Expression of a dominant negative form of TuKv2 eliminated the native delayed rectifier K+ currents, indicating that the entire delayed rectifier K+ current of the neuronal blastomere is exclusively encoded by TuKv2. TuKv2 transcripts are expressed more broadly than Na+ channel transcripts, which are restricted to the neuronal lineages. There is also a temporal mismatch in the expression of TuKv2 transcript and the K+ current; TuKv2 transcripts are present throughout development, whereas delayed rectifier K+ currents only appear after the tailbud stage, suggesting that the functional expression of the TuKv2 transcript is suppressed during the early embryonic stages. To test if this suppression occurs by a mechanism specific to the TuKv2 channel protein, an ascidian Shaker-related gene, TuKv1, was misexpressed in neural blastomeres. A TuKv1-encoded current was expressed earlier than the TuKv2 current. Furthermore, the introduction of the TuKv2-expressing plasmid into noninduced cells did not lead to the current expression. These results raise the possibility that the expression of TuKv2 is post-transcriptionally controlled through a mechanism that is dependent on neural induction.
  • K Nakajo, L Chen, Y Okamura
    JOURNAL OF PHYSIOLOGY-LONDON 515 (3) 695 - 710 0022-3751 1999/03 [Refereed][Not invited]
     
    1. Ascidian blastomeres of muscle lineage express voltage-dependent calcium channels (VDCCs) despite isolation and cleavage arrest. Taking advantage of these large dec-eloping cells, developmental changes in functional relations between VDCC currents and intracellular Ca2+ stores were studied. 2. Inactivation of ascidian VDCCs is Ca2+ dependent, as demonstrated by two pieces of evidence: (1) a bell-shaped relationship between prepulse voltage and amplitude during the test pulse in Ca2+, but not in Ba2+ and (2) the decay kinetics of Ca2+ currents (I-Ca) obtained as the size of tail currents. 3. During replacement in the external solution of Ca2+ with Ba2+, the inward current appeared biphasic: it showed rapid decay followed by recovery and slow decay This current profile was most evident in the mixed bath solution (2% Ca2+ and 98% Ba2+, abbreviated to '2Ca/98Ba'). 4. The biphasic profile of I-2Ca/98Ba was significantly attenuated in caffeine and in ryanodine, indicating that Ca2+ release is involved in shaping the current kinetics of VDCCs. After washing out the caffeine, the biphasic pattern was reproducibly restored by depolarizing the membrane in calcium-rich solution, which is expected to refill the internal Ca2+ stores. 5. The inhibitors of endoplasmic reticulum (ER) Ca2+-ATPase (SERCAs) cyclopiazonic acid (CPA) and thapsigargin facilitated elimination of the biphasic profile with repetitive depolarization. 6. At a stage earlier than 36 h after fertilization, the biphasic profile of I-2Ca/98Ba was not observed. However, caffeine induced a remarkable decrease in the amplitude of I-2Ca/98Ba and this suppression was blocked by microinjection of the Ca2+ chelator BAPTA, showing the presence of caffeine-sensitive Ca2+ stores at this stage. 7. Electron microscopic observation shows that sarcoplasmic membranes (SR) arrange closer to the sarcolemma with maturation, suggesting that the formation of the ultrastructural machinery underlies development of the cross-coupling between VDCCs and Ca2+ stores.

Books etc

  • ガイトン生理学 原著第13版
    中條 浩一 (Joint translation第4章)
    エルゼビア・ジャパン 2018/03

MISC

Awards & Honors

  • 2014/06 自然科学研究機構 第3回若手研究者賞
     
    受賞者: 中條 浩一
  • 2010/08 日本生理学会 平成22年度 入澤宏・彩記念若手研究奨励賞日本生理学会奨励賞
     
    受賞者: 中條 浩一

Research Grants & Projects

  • 心臓におけるKCNQ1チャネル修飾サブユニットの機能解明
    日本学術振興会:科研費 基盤研究(C)
    Date (from‐to) : 2017/04 -2020/03 
    Author : 中條 浩一
  • KCNQ1カリウムチャネルの腎臓における機能と慢性腎臓病との関連の解明
    大阪腎臓バンク:腎疾患研究助成
    Date (from‐to) : 2017/04 -2018/03 
    Author : 中條 浩一
  • 心臓におけるイオンチャネル複合体形成とその制御機構の解明
    内藤記念科学振興財団:内藤記念科学奨励金(研究助成)
    Date (from‐to) : 2016 -2018 
    Author : 中條 浩一
  • ストイキオメトリーの違いを考慮したKCNQ1チャネル活性化機構の光生理学的解析
    文部科学省:科研費 新学術領域研究(研究領域提案型)
    Date (from‐to) : 2013/04 -2015/03 
    Author : 中條 浩一
  • KCNQ1-KCNE1イオンチャネル複合体の結合状態と電流キネティクスの関係
    日本学術振興会:科研費 基盤研究(C)
    Date (from‐to) : 2012/04 -2015/03 
    Author : 中條 浩一
  • ストイキオメトリーの多様性を考慮に入れた新しい心筋カリウムチャネルモデルの確立
    文部科学省:科研費 新学術領域研究(研究領域提案型)
    Date (from‐to) : 2011/04 -2013/03 
    Author : 中條 浩一
  • 一分子蛍光観察によるKCNQ1-KCNE複合体構成の状況依存的変化の検出
    日本学術振興会:科研費 若手研究(B)
    Date (from‐to) : 2010/04 -2012/03 
    Author : 中條 浩一
  • ホヤKCNQ1ホモログを用いたKCNEタンパク質によるチャネル機能修飾機構の解明
    日本学術振興会:科研費 若手研究(B)
    Date (from‐to) : 2008 -2009 
    Author : 中條 浩一
  • KCNQ1チャネルのKCNE1会合による活性化遅延機構の電気・光生理学的解析
    日本学術振興会:科研費 若手研究(B)
    Date (from‐to) : 2006 -2007 
    Author : 中條 浩一


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