| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
TRPC6 is one of major TRP isoforms in vascular tissues and acts as a nonselective cation channel associated with vascular tone generation and remodeling. In our recent study, we have found that, in addition to a number of main mechanisms contributing the activation of this channel such as diacylyglycerol and IP3-receptor/calmodulin /phosphatidylinosides interaction, the phosphorylated state of the channel by calmodulin-dependent kinase II (CaMKII) greatly influence the process of activation both before and during receptor stimulation. To elucidate the molecular basis of this regulation, we have performed the following experiments. For this purpose, we performed a mutation analysis of CAMKII consensus motifs on wild-type TRPC6 and its chimera with N-terminal (NT) and transmembrane (TM) domains of TRPC7 (T776).
Search for the CAMKII consensus motif `RXX(S/T)' identified 8 and 7 candidate sequences on the NT or TM regions of TRPC6 and T776, respectively, but not on the C-terminus. Alanine
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substitution in these sequences revealed that only the mutations T487A in wild type TRPC6 and T433A in T776 strongly attenuated Ba^<2+> influx evoked by carbachol (100μM) without affecting their cell-membrane localized expression. The critical importance of T487A for TRPC6 activation was also confirmed by patch clamp experiments, where carbachol-induced current (ITRPC6) was reduced from 9.4±2.3 to 1.2±0.4 (pA/pF, n=5) by alanine substitution. In addition, substitution of T487 with glutamine, which confers a permanently negative charge therefore, caused potentiation of the current and Ba2+ response to carbachol, with prolong deactivation after termination of receptor stimulation. On the other hand, the cell membrane localized expression of TRPC6 protein assessed by its immunofluorescence did not appreciably change by these mutations. Concidering the proposed membrane topology derived from the structural analysis of TRPC1, T487 in TRPC6 and T433 in T776 are likely located on a long intracellular stretch between the second and third TM domains (II- III loop). These results suggest, combined with the requisiteness of a putative calmodulin binding site (CIRB) for channel activation, that close spatial arrangement of CIRB and the II-III loop might allow effective phosphorylation of T487 by CAMKII. This might in turn prime and/or facilitate the TRPC6 channel gating toward 'opening', which might be greatly affected by the charged state of T487 and negatively charged phospholipids in its close vicinity.
In the course of molecular elucidation of CaMKII-mediated regulation, we laso noticed that one of consensus phosphorylation motif T69 is common with that of protein kinase G (PKG), Since PKG is an important target of the ubiquitous physiological vasorelaxant nitric oxide (NO) and also since vasoconstrictor-induced Ca2+ entry is known to be strongly inhibited by activation of NO/cGMP/PKG pathway, we explored the potential role of this system in regulating TRPC6 channel activity. The magnitude of ITRPC6 was greatly inhibited by pretreatment with a NO donor, S-nitrosoacetyl penicillamine (SNAP) (by 70% at 100 μ M) in a voltage-independent manner, but cell-surface expression of TRPC6 protein was not appreciably reduced. Similar extent of inhibition was produced by a membrane-permeable analogue of cGMP 8-bromo cGMP (8-Br-cGMP; 100 g M). Both the inhibitory effects of SNAP and 8-Br-cGMP were nearly abolished by simultaneous administration of the PKG inhibitor KT5823 (10 μ M), PKG-specific inhibitory peptide DT-3 or alanine substitution for T69. These results suggest that PKG-mediated phosphorylation is another powerful mechanism to control TRPC6 channel activity, which may in situ operate as a tonic negative feedback via NO produced in adjacent endothelial cells or migrating inflammatory cells. Less
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