Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref. 8). When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.

Abstract. Centromere protein-F (CENP-F) is a mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner,

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by ␤-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells. mitochondria ͉ permeability transition ͉ polyphosphate ͉ ␤-amyloid peptide ͉ necrosis T he chemical and physical properties of polyphosphate (polyP), including its high negative charge and its ability to form complexes with Ca 2ϩ and to form high energy bonds, underlie its potential to play an important role in cell metabolism. Significant amounts of polyP have been found in bacteria and in lower eukaryotes. In those organisms, it provides energy storage and a reserve pool of inorganic phosphate, participates in regulation of gene expression, protects cells from the toxicity of heavy metals by forming complexes with them, and participates in channel formation through assembly into complexes with Ca 2ϩ and polyhydroxybutyrate (PHB) (polyP/Ca 2ϩ /PHB complex) (1, 2) and possibly through interaction with channelforming proteins (3).PolyP has also been found in all higher eukaryotic organisms tested, where it is localized in various subcellular compartments, including mitochondria (4). Furthermore, mitochondrial polyP can form polyP/Ca 2ϩ /PHB complexes (5) with ion-conducting properties similar to those of native mitochondrial permeability transition pore (mPTP) (6). mPTP opening or formation in the mitochondrial inner membrane is believed to underlie the Ca 2ϩ -induced permeability transition (PT), a phenomenon that causes inner membrane depolarization and disruption of ATP synthesis and plays a central role during various types of necrotic and apoptotic cell death (7). The molecular composition of the conducting pathway of mPTP is currently not well defined.Recently, we have raised the possibility that, in vivo, the polyP/ Ca 2ϩ /PHB complex might comprise the ion-conducting part of the mPTP complex (6). If so, mitochondrial polyP should be essential for mPTP opening/formation. Here, we examine the involvement of polyP in normal mitochondrial function and in PT development during stress. To this end, we specifically reduced levels of mitocho...

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca 2؉ Ryanodine receptors (RyRs) 1 are members of a superfamily of intracellular Ca 2ϩ channels that include the inositol 1,4,5-trisphosphate receptors (IP 3 Rs). These channels play an essential role in intracellular Ca 2ϩ signaling by virtue of releasing Ca 2ϩ from the lumen of sarco(endo)plasmic reticulum to the cytosol of muscle and non-muscle cells (1, 2).RyR is a homotetrameric structure composed of four identical subunits, each having ϳ5000 amino acids. Sequence analysis reveals that one-fifth of the COOH terminus of the molecule is likely to form the channel conducting pore. The remaining ϳ4000 amino acid residues apparently constitute the cytoplasmic "foot" domain (3-7). A truncated RyR in which the foot domain has been deleted has been shown to function as a Ca 2ϩ release channel. The truncated RyR channel was still regulated by Ca 2ϩ , was modified by ryanodine, and exhibited a single channel conductance similar to that of the full-length RyR (8). These studies indicate that the sites for Ca 2ϩ activation and ryanodine binding, and the ion conduction pathway are located within the COOH-terminal ϳ1000 amino acid residues. A glutamate residue located in the putative transmembrane sequence M2 has recently been identified as the Ca 2ϩ sensor of RyR (9). The locations of the ryanodine binding site and the pore-forming segment of RyR, however, have yet to be defined.RyRs and IP 3 Rs share some sequence homology, in particular in the COOH-terminal channel-forming domain (10). Considering their sequence homology and similar conduction properties (11-13), RyRs and IP 3 Rs are likely to share similar structural features in the channel pore. A hydrophobic region between the M5 and M6 transmembrane sequences of the mouse type 1 IP 3 R has been proposed to be the pore-forming region (14). The equivalent region in RyR, corresponding to the M9 transmembrane sequence proposed by Zorzato et al. (15), is also hydrophobic. Sequence alignment of these regions reveals a GXRXGGGXGD motif that can be found in all known RyRs and IP 3 Rs (Fig. 1). To investigate its role in RyR function, we have introduced point mutations into this highly conserved region and examined the functional consequences of these point mutations. Our data indicate that this region is critical for ryanodine binding and ion conduction and probably constitutes the pore-forming segment of RyRs. EXPERIMENTAL PROCEDURES Materials-Ryanodine was obtained from Calbiochem. [3 H]Ryanodine was from NEN Life Science Products. Monoclonal antibody 34C was a generous gift from Dr. John L. Sutko (16).Cloning of the Mouse Cardiac RyR cDNA-Total RNA from mouse heart tissue, isolated by the method of Chomczynski and Sacchi (17), was used to generate firs...

Voltage-gated sodium channels are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Potassium channels that are inhibited by internal ATP (KATP channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K ATP channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K ATP channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K ATP channel isoforms suggests that this pathway may have wide significance for the physiological regulation of KATP channels in various tissues and organelles. P otassium channels that are inhibited by ATP (K ATP channels) consist of a heterooctamer of four sulfonylurea receptor (SURx) and four inwardly rectifying K ϩ channel (Kir6.x) subunits (1-5). The SUR is a member of the ATP-binding cassette (ABC) family of proteins and acts as a regulatory subunit, conferring ADP sensitivity and the distinctive pharmacological characteristics on the K ATP channel complex (1-6). In contrast, the Kir6.x subunit forms the pore of the channel and mediates the defining ATP-dependent inhibition of K ATP channels (6). Protein kinase-catalyzed phosphorylation is an important mechanism by which the activity of ion channels, including the K ATP channel, can be controlled (7-9). For instance, another ABC protein ion channel, the cystic fibrosis transmembrane conductance regulator, is regulated by cAMP-dependent protein kinase-mediated phosphorylation, which, itself, may be permissively regulated by protein kinase C (PKC) (8, 10, 11). In addition, mounting evidence suggests the importance of PKC in activating K ATP channels during both the protective mechanism of ischemic preconditioning (12, 13) and in regulating insulin secretion (14), although the site(s) and mechanism of action of PKC-mediated phosphorylation events have not been described. Therefore, we sought to determine: (i) the functional effects of PKC on the K ATP channel, (ii) whether the action of PKC is mediated via the SUR or Kir6.2 subunit, and (iii) the identity of specific amino acid residue(s) phosphorylated by PKC. Materials and MethodsCell Culture and Transfection. tsA201 cells (an SV40-transformed variant of the HEK293 human embryonic kidney cell line) were maintained in DMEM supplemented with 10 mM glucose͞2 mM L-glutamine͞10% FCS͞0.1% penicillin/streptomycin at 37°C (10% CO 2 ). Cells were plated at 30-40% confluence on 35-...

L-type, voltage-gated Ca2؉ channels (Ca L ) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle Ca L channel isoforms are acutely potentiated following ␣5␤1 integrin activation. Only the ␣ 1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by ␣5␤1 integrin requires phosphorylation of ␣ 1C C-terminal residues Ser 1901 and Tyr 2122 . These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following ␣5␤1 integrin activation, native Ca L channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of Ca L channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.Voltage-gated calcium channels play critical roles in the regulation of calcium entry across the plasma membranes of excitable cells. L-type calcium channels (Ca L ), 5 which are highly expressed in brain and muscle, are heteromeric transmembrane proteins composed of a poreforming ␣ 1C (Cav1.2) subunit along with accessory ␤, ␣ 2 , ␦, and sometimes ␥ subunits (1, 2). The ␣ 1C subunit contains four highly conserved repeat regions with 24 membrane-spanning domains, in addition to a variable length N terminus and relatively long, intracellular C terminus. The three ␣ 1C isoforms (neuronal, Cav1.2c; smooth muscle, Cav1.2b; cardiac, Cav1.2a) exhibit significant sequence differences in their N and C termini but all are regulated by intracellular kinases in ways that uniquely determine calcium entry and cell excitability.The regulation of Ca L channels by serine-threonine kinases has been extensively investigated. PKG phosphorylates a conserved serine reside in the cytoplasmic I-II linker (3) of all three ␣ 1C isoforms, leading to inhibition of current. PKC phosphorylates N-terminal threonine residues in cardiac and smooth muscle isoforms (4 -6) leading in most cases to potentiation of current. PKA phosphorylates all three ␣ 1C isoforms at a conserved C-terminal serine (Ser 1901 in Cav1.2c; Ser 1928 in Cav1.2a), thereby mediating ␤-adrenergic potentiation of the calcium current in cardiac myocytes and neurons (7-9). PKA also regulates ␣ 1C in smooth muscle, but the functional consequences on calcium current are complicated by crossover activation of PKG, which is expressed at high levels in that tissue (10).We recently demonstrated that Ca L currents in vascular smooth muscle (VSM) are acutely regulated by the integrin class of cel...