Minoru Wakamori

Ca2+–calmodulin‐dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor‐activated Ca2+ influx in vertebrate cells. We previously reported the involvement of intracellular Ca2+ in the receptor‐mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca2+, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca2+ entry through TRPC5 channels, induced upon stimulation of the G‐protein‐coupled ATP receptor, was abolished by treatment with W‐13, an inhibitor of calmodulin. ML‐9 and wortmannin, inhibitors of Ca2+–calmodulin‐dependent myosin light chain kinase (MLCK), and the expression of a dominant‐negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML‐9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole‐cell patch‐clamp technique was inhibited by ML‐9, whereas TRPC5 channel activity observed in the cell‐excised, inside‐out patch was unaffected by ML‐9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML‐9 in HEK293 cells. These findings strongly suggest that intracellular Ca2+–calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.

Properties of native P2X receptors in large multipolar neurons dissociated from rat hypothalamic arcuate nucleus

ATP, the ligand of P2X receptors, is a candidate of neurotransmitter or co-transmitter in the peripheral and the central nervous systems. Anatomical studies have revealed the wide distribution of P2X receptors in the brain. So far, P2X-mediated small synaptic responses have been recorded in some brain regions. To determine the physiological significance of postsynaptic ATP receptors in the brain, we have investigated the P2X responses in rat dissociated hypothalamic arcuate neurons by using the patch-clamp technique. ATP evoked inward currents in a concentration-dependent manner (EC(50)=42 microM) at a holding potential of -70 mV. The current-voltage relationship showed a marked inward rectification starting around -10 mV. Although neither 300 microM alphabeta-methylene-ATP nor 300 microM betagamma-methylene-ATP induced any currents, 100 microM ATPgammaS and 100 microM 2-methylthio-ATP evoked inward currents of which amplitude was about 60% of the control currents evoked by 100 microM ATP. PPADS, one of P2 receptor antagonists, inhibited the ATP-evoked currents in a time- and a concentration-dependent manners (IC(50)=19 microM at 2 min). Permeant Ca(2+) inhibited the ATP-evoked currents in the range of millimolars (IC(50)=7 mM); however, Cd(2+) (1-300 microM), a broad cation channel blocker, facilitated the currents with slow off-response. Zn(2+) in the range of 1-100 microM facilitated the currents whereas Zn(2+) at the concentrations over 100 microM inhibited the currents. These observations suggest that functional P2X receptors are expressed in the hypothalamic arcuate nucleus. The most likely subunit combinations of the P2X receptors are P2X(2)-homomultimer and P2X(2)/P2X(6)-heteromultimer.

Limited intercellular spread of spontaneous Ca2+ signals via gap junctions between mouse chromaffin cells in situ

Using a confocal laser-scanning microscope, we measured the changes in the cytosolic Ca(2+) concentration ([Ca]i) of chromaffin cells on adrenal slice preparations of mouse. The spontaneous fluctuations of [Ca]i were often observed in situ, as reported in isolated rat and bovine cells. Intriguingly, the spontaneous [Ca]i changes in one cell were often transmitted to one or two adjacent cells, and the synchronized [Ca]i changes were often observed in two adjacent cells, both of which failed to respond to ATP. The synchronized [Ca]i changes between two cells were much less frequently observed in rat than in mouse adrenals. The spontaneous [Ca]i changes were also synchronized between closely touching mouse chromaffin cells in culture. These results suggest that the spread of Ca(2+) signaling is mediated by low-conductance intercellular coupling.

Nicotinic ACh receptors in area postrema neurons of immature rat brain

The electrophysiological properties of nicotinic ACh receptors (nAChR) were investigated in acutely dissociated area postrema (AP) neurons of the immature rat brain using the whole-cell patch-clamp recording method. ACh induced a transient inward current exhibiting a strong inward rectification. The ACh response was mimicked by nicotine and cytisine, and was inhibited by nAChR antagonists, but not by 10(-7) M atropine. Muscarinic AChR agonists did not induce any current. We confirmed the Ca2+ permeability of nAChR. These results indicate the presence of nAChR on AP neurons, and suggest that the activation of nAChR play important roles in cardiovascular functions in rats.