Takahiro Ishii

A family of metabotropic glutamate receptors.

Three cDNA clones, mGluR2, mGluR3, and mGluR4, were isolated from a rat brain cDNA library by cross-hybridization with the cDNA for a metabotropic glutamate receptor (mGluR1). The cloned receptors show considerable sequence similarity with mGluR1 and possess a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR2 is expressed in some particular neuronal cells different from those expressing mGluR1 and mediates an efficient inhibition of forskolin-stimulated cAMP formation in cDNA-transfected cells. The mGluRs thus form a novel family of G protein-coupled receptors that differ in their signal transduction and expression patterns.

Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing

We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.

Molecular Characterization of the Hyperpolarization-activated Cation Channel in Rabbit Heart Sinoatrial Node

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (I for I h) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brainI f cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1–3. The transmembrane region of HAC4 was most homologous to partially cloned mouse I f BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at −87.2 ± 2.8 mV under control conditions and at −64.4 ± 2.6 mV in the presence of intracellular 0.3 mm cAMP. The reversal potential was −34.2 ± 0.9 mV in 140 mm Na+ o and 5 mm K+ o versus 10 mm Na+ i and 145 mmK+ i . These results indicate that HAC4 formsI f in rabbit heart SA node.

Axonal site of spike initiation enhances auditory coincidence detection

Neurons initiate spikes in the axon initial segment or at the first node in the axon. However, it is not yet understood how the site of spike initiation affects neuronal activity and function. In nucleus laminaris of birds, neurons behave as coincidence detectors for sound source localization and encode interaural time differences (ITDs) separately at each characteristic frequency (CF). Here we show, in nucleus laminaris of the chick, that the site of spike initiation in the axon is arranged at a distance from the soma, so as to achieve the highest ITD sensitivity at each CF. Na+ channels were not found in the soma of high-CF (2.5–3.3 kHz) and middle-CF (1.0–2.5 kHz) neurons but were clustered within a short segment of the axon separated by 20–50 μm from the soma; in low-CF (0.4–1.0 kHz) neurons they were clustered in a longer stretch of the axon closer to the soma. Thus, neurons initiate spikes at a more remote site as the CF of neurons increases. Consequently, the somatic amplitudes of both orthodromic and antidromic spikes were small in high-CF and middle-CF neurons and were large in low-CF neurons. Computer simulation showed that the geometry of the initiation site was optimized to reduce the threshold of spike generation and to increase the ITD sensitivity at each CF. Especially in high-CF neurons, a distant localization of the spike initiation site improved the ITD sensitivity because of electrical isolation of the initiation site from the soma and dendrites, and because of reduction of Na+-channel inactivation by attenuating the temporal summation of synaptic potentials through the low-pass filtering along the axon.

Determinants of activation kinetics in mammalian hyperpolarization-activated cation channels

1 The structural basis for the different activation kinetics of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels was investigated with the whole‐cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS−7). 2 The activation time constant of HCN4 was about 40‐fold longer than that of HCN1 when compared at −100 mV. 3 In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1‐S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide‐binding domain (CNBD) also significantly affected the activation kinetics. 4 The S1 domain and S1‐S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2‐fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1‐S2 region of HCN1 by that of HCN4 resulted in a 10− to 20‐fold slowing. 5 The results indicate that S1 and S1‐S2, and S6‐CNBD are the crucial components for the activation gating of HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated channels and T-type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes.

Regeneration of cardiac pacemakers is an important target of cardiac regeneration. Previously, we developed a novel embryonic stem (ES) cell differentiation system that could trace cardiovascular differentiation processes at the cellular level. In the present study, we examine expressions and functions of ion channels in ES cell‐derived cardiomyocytes during their differentiation and identify ion channels that confer their automaticity. ES cell‐derived Flk1+ mesoderm cells give rise to spontaneously beating cardiomyocytes on OP9 stroma cells. Spontaneously beating colonies observed at day 9.5 of Flk1+ cell culture (Flk‐d9.5) were significantly decreased at Flk‐d23.5. Expressions of ion channels in pacemaker cells hyperpolarization‐activated cyclic nucleotide‐gated (HCN)1 and ‐4 and voltage‐gated calcium channel (Cav)3.1 and ‐3.2 were significantly decreased in purified cardiomyocytes at Flk‐d23.5 compared with at Flk‐d9.5, whereas expression of an atrial and ventricular ion channel, inward rectifier potassium channel (Kir)2.1, did not change. Blockade of HCNs and Cav ion channels significantly inhibited beating rates of cardiomyocyte colonies. Electrophysiological studies demonstrated that spontaneously beating cardiomyocytes at Flk‐d9.5 showed almost similar features to those of the native mouse sinoatrial node except for relatively deep maximal diastolic potential and faster maximal upstroke velocity. Although ∼60% of myocytes at Flk‐d23.5 revealed almost the same properties as those at Flk‐d9.5, ∼40% of myocytes showed loss of HCN and decreased Cav3 currents and ceased spontaneous beating, with no remarkable increase of Kir2.1. Thus, HCN and Cav3 ion channels should be responsible for the maintenance of automaticity in ES cell‐derived cardiomyocytes. Controlled regulation of these ion channels should be required to generate complete biological pacemakers.

Molecular characterization of NMDA and metabotropic glutamate receptors.

Our molecular studies have revealed the existence of a large number of different subunits or subtypes for the NMDA and metabotropic glutamate receptors. The individual receptors show functional variabilities and distinct expression patterns in the CNS. The NMDA receptors belong to the ligand-gated ion channel family and consist of a key subunit NMDAR1 and four accessory subunits NMDAR2A-NMDAR2D. The combination of NMDAR1 and NMDAR2 in heteromeric configurations potentiates glutamate response and produces a functional variability. All the NMDAR subunits have an asparagine residue at the corresponding position of the second transmembrane segments, and these residues are thought to be responsible for controlling Ca2+ permeation and the channel blockade by Mg2+ and cationic channel blockers. Individual NMDAR subunit mRNAs are different in their expression patterns during development and in the adult brain. The mGluR family consists of at least six different subtypes. These subtypes are divided into three subgroups according to their sequence similarities, signal transduction mechanisms, and pharmacological properties. Although their physiological roles largely remain to be elucidated, the retinal L-AP4-sensitive mGluR may have a specific function that mediates excitatory neurotransmission in the visual system. It is thus undoubtedly important to investigate specific functions of different combinations of the NMDA receptor subunits and different subtypes of mGluRs and to explore the molecular mechanisms of glutamate receptor-mediated neuronal plasticity and neurotoxicity.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Cation Channels Regulate Auditory Coincidence Detection in Nucleus Laminaris of the Chick

Coincidence detection of bilateral acoustic signals in nucleus laminaris (NL) is the first step in azimuthal sound source localization in birds. Here, we demonstrate graded expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels along the tonotopic axis of NL and its role in the regulation of coincidence detection. Expression of HCN1 and HCN2, but not HCN3 or HCN4, was detected in NL. Based on measurement of both subtype mRNA and protein, HCN1 varied along the tonotopic axis and was minimal in high-characteristic frequency (CF) neurons. In contrast, HCN2 was evenly distributed. The resting conductance was larger and the steady-state activation curve of Ih was more positive in neurons of middle to low CF than those of high CF, consistent with the predominance of HCN1 channels in these neurons. Application of 8-Br-cAMP or noradrenaline generated a depolarizing shift of the Ih voltage activation curve. This shift was larger in neurons of high CF than in those of middle CF. The shift in the activation voltage of Ih depolarized the resting membrane, accelerated the EPSP time course, and significantly improved the coincidence detection in neurons of high CF, suggesting that Ih may improve the localization of sound sources.

SITE-DIRECTED MUTAGENESIS

Publisher Summary Ion channels are ubiquitous membrane proteins that are centrally important to the existence of all cells. Indeed, a sequence with remarkable homology to a mammalian K + channel resides in the Escherichia coli genome. In the mammalian CNS, each cell may possess a unique complement of ion channels, a fingerprint of the personality of the cell. Prior to cloning, the unprecedented array of information from electrophysiologic recordings motivated detailed speculation concerning the underlying molecular architecture of ion channel proteins. Thus, Armstrong used recordings coupled with biochemical approaches to infer the mechanism of channel inactivation, and proposed the model of a ball and chain inactivation particle for Na + channels. The concepts of a specialized segment residing in the membrane electric field that mediates the responses to voltage changes, a hydrophilic core for ion translocation surrounded by a hydrophobic interface with the lipid bilayer, and amino acids that determine the selectivity for different ions as well as the rate at which they flux across the membrane were incorporated into ever more detailed extrapolations of possible ion channel structures. This chapter discusses the most widely employed and useful techniques for site-directed mutagenesis of ion channels. It presents some background on the evolution of these techniques and highlights major conceptual advances. Three basic protocols are described in detail, and a discussion of their relative merits and drawbacks is presented. An appendix is included with buffer compositions and some detailed supporting methods.

Inhibition of the human intermediate conductance Ca2+-activated K+ channel, hIK1, by volatile anesthetics

Ca(2+)-activated K(+) channels (K(Ca)) regulate a wide variety of cellular functions by coupling intracellular Ca(2+) concentration to membrane potential. There are three major groups of K(Ca) classified by their unit conductances: large (BK), intermediate (IK), and small (SK) conductance of channels. BK channel is gated by combined influences of Ca(2+) and voltage, while IK and SK channels are gated solely by Ca(2+). Volatile anesthetics inhibit BK channel activity by interfering with the Ca(2+) gating mechanism. However, the effects of anesthetics on IK and SK channels are unknown. Using cloned IK and SK channels, hIK1 and hSK1-3, respectively, we found that the currents of hIK1 were inhibited rapidly and reversibly by volatile anesthetics, whereas those of SK channels were not affected. The IC(50) values of the volatile anesthetics, halothane, sevoflurane, enflurane, and isoflurane for hIK1 inhibition were 0.69, 0.42, 1.01 and 1.03 mM, respectively, and were in the clinically used concentration range. In contrast to BK channel, halothane inhibition of hIK1 currents was independent of Ca(2+) concentration, suggesting that Ca(2+) gating mechanism is not involved. These results demonstrate that volatile anesthetics, such as halothane, enflurane, isoflurane, and sevoflurane, affect BK, IK, and SK channels in distinct ways.