The effects of RGS4 on the voltage‐dependent relaxation of G protein‐gated K+ (KG) channels were examined by heterologous expression in Xenopus oocytes. While the relaxation kinetics was unaffected by the acetylcholine concentration ([ACh]) in the absence of RGS4, it became dependent on [ACh] when RGS4 was co‐expressed. Kinetic analyses indicated that RGS4 confers to the KG channel a voltage‐independent inhibitory gating mechanism, which was attenuated by ACh in a concentration‐dependent fashion. In vitro biochemical studies showed that RGS4 could bind to the protein complex containing KG channel subunits. Since the native cardiac KG channel exhibited similar agonist‐dependent relaxation kinetics to that mediated by RGS4, it is suggested that KG channel gating is a novel physiological target of RGS protein‐mediated regulation.

In native cardiac myocytes, there is a time dependence to the G protein‐gated inwardly rectifying K+ (KG) channel current during voltage steps that accelerates as the concentration of acetylcholine is increased. This phenomenon has been called ‘relaxation’ and is not reproduced in the reconstituted Kir3.1/Kir3.4 channel in Xenopus oocytes. We have shown that RGS4, a regulator of G protein signalling, restores relaxation to the reconstituted Kir3.1/Kir3.4 channel. In this study, we examined the mechanism of this phenomenon by expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and mutants of RGS4 in Xenopus oocytes. RGS4 restored relaxation to KG channels activated by the pertussis toxin (PTX)‐sensitive G protein‐coupled m2‐muscarinic receptor but not to those activated by the Gs protein‐coupled β2‐adrenergic receptor. RGS4 induced relaxation not only in heteromeric KG channels composed of Kir3.1 and Kir3.4 but also in homomeric assemblies of either an active mutant of Kir3.1 (Kir3.1/F137S) or an isoform of Kir3.2 (Kir3.2d). Truncation mutants of RGS4 showed that the RGS domain itself was essential to reproduce the effect of wild‐type RGS4 on the KG channel. The mutation of residues in the RGS domain which interact with the α subunit of the G protein (Gα) impaired the effect of RGS4. This study therefore shows that interaction between the RGS domain and PTX‐sensitive Gα subunits mediates the effect of RGS4 on the agonist concentration‐dependent relaxation of KG channels.

We examined protein kinase C (PKC) activity in Ca2-dependent PKC (Ca2-dependent PKC activities) and Ca2-independent PKC (Ca2-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca2~-dependent and Ca2-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca2~-independentPKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-, was used in the assay; whereas Ca2~-independentPKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-6, was not significantly reduced in AD. Further studies on the protein levels of Ca2~-independentPKC-6, PKC-e, and PKC-ĩn AD brain revealed reduction of the PKClevel in both cytosolic and membranous fractions, although PKC-6 and PKC-levels were not changed. These findings indicated that Ca2-dependent and Ca2~-independent PKC are changed in AD, and that among Ca2~independent PKC isozymes, the alteration of PKC-is a specific event in AD brain, suggesting its crucial role in AD pathophysiology. Key Words: Alzheimer's disease-Ca2 -dependent protein kinase C-Ca 2k-independent protein kinase C-Protein kinase C-c-Brain.

No abstract

A novel antidiabetic agent, nateglinide, is a D-phenylalanine derivative lacking either a sulfonylurea or benzamido moiety. We examined with the patch-clamp method the effect of nateglinide on recombinant ATP-sensitive K ϩ (K ATP ) channels expressed in human embryonic kidney 293T cells transfected with a Kir6.2 subunit and either of a sulfonylurea receptor (SUR) 1, SUR2A, and SUR2B. In inside-out patches, nateglinide reversibly inhibited the spontaneous openings of all three types of SUR/Kir6.2 channels. Nateglinide inhibited SUR1/Kir6.2 channels with high and low affinities (K i ϭ 75 nM and 114 M) but SUR2A/Kir6.2 and SUR2B/Kir6.2 channels only with low affinity (K i ϭ 105 and 111 M, respectively). Nateglinide inhibited the K ATP current mediated by Kir6.2 lacking C-terminal 26 amino acids only with low affinity (K i ϭ 290 M) in the absence of SUR. Replacement of serine at position 1237 of SUR1 to tyrosine [SUR1(S1237Y)] specifically abolished the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide. MgADP or MgUDP (100 M) augmented the inhibitory effect of nateglinide on SUR1/Kir6.2 but not SUR1(S1237Y)/Kir6.2 or SUR2A/Kir6.2 channels. This augmenting effect of MgADP was also observed with the SUR1/Kir6.2(K185Q) channel, which was not inhibited by MgADP, but not with the SUR1(K1384A)/Kir6.2 channel, which was not activated by MgADP. These results indicate that therapeutic concentrations of nateglinide (ϳ10 M) may selectively inhibit pancreatic type SUR1/Kir6.2 channels through SUR1, especially when the channel is activated by intracellular MgADP, even though the agent does not contain either a sulfonylurea or benzamido moiety. ATP-sensitive Kϩ (K ATP ) channels are inhibited by intracellular ATP and activated by ADP and thus provide a link between the cellular metabolic state and excitability (Ashcroft, 1988;Terzic et al., 1995). These channels are associated with such cellular functions as insulin secretion, cardiac preconditioning, vasodilatation, and neuroprotection (Ashcroft, 1988;Terzic et al., 1995;Nichols et al., 1996;Quayle et al., 1997;Yamada et al., 2001;Miki et al., 2002). K ATP channels are composed of an ATP-binding cassette protein, sulfonylurea receptor (SUR), and an inwardly rectifying K ϩ channel (Kir) subunit, Kir6.0 (Aguilar-Bryan et al., 1995;Inagaki et al., 1995Inagaki et al., , 1996Inagaki et al., , 1997Sakura et al., 1995;Clement et al., 1997;Shyng and Nichols, 1997). Detailed functional analyses of K ATP channels composed of Kir6.0 and either of three types of SUR (SUR1, SUR2A, and SUR2B) indicate that SUR1, SUR2A, and SUR2B represent pancreatic, cardiac, and vascular smooth muscle types of SUR, respectively (AguilarBryan et al