Masayuki Mori

Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells

We investigated, by using the patch clamp technique, Ca2+‐mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor‐operated Ca2+ entry channels. With nystatin‐perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 μm)‐activated TRPC6 currents (ITRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2o+) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2o+ solely inhibited TRPC7 currents (ITRPC7). Vigorous buffering of intracellular Ca2+ (Ca2i+) under conventional whole‐cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2o+, disclosing fast potentiation (EC50: ∼0.4 mm) and inhibition (IC50: ∼4 mm) of ITRPC6 and fast inhibition (IC50: ∼0.4 mm) of ITRPC7. This inhibition of ITRPC6 and ITRPC7 seems to be associated with voltage‐dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of ITRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of ITRPC6 or its acceleration by Ca2o+ probably involves phosphorylation by calmodulin (CaM)‐dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 μm), coexpression of Ca2+ ‐insesentive mutant CaM, and intracellular perfusion of the non‐hydrolysable ATP analogue AMP‐PNP and a CaMKII‐specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh‐activated TRPC7 channel activity was concentration‐dependently suppressed by nanomolar Ca2i+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of ITRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+−CaM‐dependent and ‐independent mechanisms.

Nitric oxide-cGMP-protein kinase G pathway negatively regulates vascular transient receptor potential channel TRPC6.

We investigated the inhibitory role of the nitric oxide (NO)–cGMP–protein kinase G (PKG) pathway on receptor‐activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by ∼70%) by a NO donor SNAP (100 μm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 μm) or after G‐protein activation with intracellular perfusion of GTPγS (100 μm). A similar extent of suppression was also observed with a membrane‐permeable analogue of cGMP, 8Br‐cGMP (100 μm). The inhibitory effects of SNAP and 8Br‐cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br‐cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside‐out configuration in a PKG‐dependent manner. SNAP‐induced PKG activation stimulated the incorporation of 32P into wild‐type and S321A‐mutant TRPC6 proteins immunoprecipitated by TRPC6‐specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br‐cGMP strongly suppressed TRPC6‐like cation currents and membrane depolarization evoked by Arg8‐vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO–cGMP–PKG pathway, probably via T69 phosphorylation of the N‐terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

A human Dravet syndrome model from patient induced pluripotent stem cells

BackgroundDravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Nav1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome’s human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs.ResultsWe generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected.ConclusionsOur results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies.

DNA segments mapped by reciprocal use of microsatellite primers between mouse and rat.

Rat microsatellite primers were used for detection of homologous DNA segments in the mouse species (Mus laboratorius, Mus musculus musculus, and Mus spretus). Twenty five (16.3%) of 153 rat primer pairs amplified specific DNA segments, when genomic DNA of mice was used as a template in the polymerase chain reaction (PCR). Size variation among inbred strains of mice was found for 13 DNA segments (8.5%). Eight out of the 13 polymorphic DNA segments were mapped to a particular chromosome with two sets of recombinant inbred strains, AKXL or BXD. Similarly, mouse microsatellite primers were used for detection of homologous DNA segments in rats (Rattus norvegicus). Twenty (12.0%) of 166 primer pairs amplified specific DNA segments from rat genome. Size variation among inbred strains of rats was found for seven DNA segments (4.2%). Eleven of these 20 DNA segments were mapped with a rat x mouse somatic cell hybrid clone panel and/or linkage analysis by use of backcross progeny. Our results suggest that the mapped DNA segments are really homologs between mouse and rat. These polymorphic DNA segments are useful genetic markers.

Chromosomal assignments of 17 structural genes and 11 related DNA fragments in rats (Rattus norvegicus) by Southern blot analysis of rat × mouse somatic cell hybrid clones

DNA from 18 rat x mouse somatic cell hybrid clones, which segregated individual rat chromosomes, was analyzed by Southern blot for chromosomal gene assignments. Through the use of 17 DNA probes cloned from 7 rat genes, A2M, ATP1A1, ATP1A2, ATP1A3, B2M, GSTP, and SMST; 5 mouse genes, Ncam, Ngfg, Pim-1, Tcp-1, and Trp53; and 5 human genes, MBP, MYB, NEFM, SCN2A, and TCRGC1, 17 structural genes including 15 newly assigned genes and 11 related DNA fragments were assigned to particular rat chromosomes. Syntenic conservation of the genes among rats, mice, and humans is discussed.

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels

Phosphatidylinositol 4,5-bisphosphate has a direct role in regulating receptor-operated TRPC channel activation and inactivation.

A self‐limiting regulation of vasoconstrictor‐activated TRPC3/C6/C7 channels coupled to PI(4,5)P2‐diacylglycerol signalling

•  From brain to digestive tract, electro‐chemical signals are broadly utilized to control the activity of the organs; however, the formation of such signals is very varied in each cell and still unknown in many cells. •  In this study, we found a novel mechanism for forming an electrical signal, produced by channels of the transient receptor potential canonical (TRPC) family of channels, which allow the permeation of ions such as sodium and calcium and are opened by the actions of hormones such as adrenaline and noradrenaline. •  Such hormones can activate an enzyme (phospholipase C) by which PI(4,5)P2, a member of the membrane lipid ‘phosphoinositide’, is degraded: the degradation of PI(4,5)P2 to produce an agonist (diacylglycerol) involved in the opening of TRPC channels, while the degradation itself is surprisingly critical to the closing of these channels. •  As a result of such a self‐limiting effect via membrane lipid degradation, TRPC channels can produce a unique electro‐chemical signal which is tightly bound to the arrangement of membrane lipid and hormones. •  Differential sensitivity for PIP2 of TRPC

Restriction fragment length polymorphisms of the angiotensinogen gene in inbred rat strains and mapping of the gene on chromosome 19q

Using a cDNA probe of the rat angiotensinogen gene (ANG), restriction fragment length polymorphisms (RFLPs) were detected in inbred rat strains with the restriction enzymes HindIII, PstI, and PvuII. Three alleles of ANG were almost equally distributed in 11 inbred strains. In two sets of backcross progeny originating from parental strains with different alleles, no close linkage was found between the ANG locus and 17 other loci tested. In situ hybridization, however, allowed assignment of the gene to chromosome 19q. The RFLPs of the angiotensinogen gene, therefore, can be considered useful as markers of rat chromosome 19.

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe.

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.

Stable expression and characterization of human PN1 and PN3 sodium channels.

Nociceptive transduction in inflammatory and neuropathic pain involves peripherally expressed voltage-gated sodium channels, such as tetrodotoxin (TTX)-sensitive PN1 and TTX-resistant PN3. We generated recombinant cell lines stably expressing the human PN1 and PN3 sodium channels in Chinese hamster ovary (CHO) cells using inducible expression vectors. The PN1 and PN3 cDNAs were isolated from human adrenal gland and heart poly(A)+ RNAs, respectively. The recombinant human PN1 currents exhibited rapid activation and inactivation kinetics and were blocked by TTX with a half-maximal inhibitory concentration (IC50) of 32.6 nM. The human PN3 channel expressed in stable transfectants showed TTX-resistant inward currents with slow inactivation kinetics. The IC50 value for TTX was 73.3 microM. The voltage-dependence of activation of the PN3 channel was shifted to the depolarizing direction, compared to that of the PN1 channel. Lidocaine and mexiletine exhibited tonic and use-dependent block of PN1 and PN3 channels. The PN1 channel was more susceptible to inhibition by mexiletine than PN3. These results suggest that stable transfectants expressing the human PN1 and PN3 sodium channels will be useful tools to define subtype selectivity for sodium channel blockers.