David L. Armstrong

Orai proteins interact with TRPC channels and confer responsiveness to store depletion

The TRPC (C-type transient receptor potential) class of ion channels has been hypothesized to participate in store-operated Ca2+ entry (SOCE). Recently, however, STIM1 and Orai1 proteins have been proposed to form SOCE channels. Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored. Here we show that Orai1 physically interacts with the N and C termini of TRPC3 and TRPC6, and that in cells overexpressing either TRPC3 or TRPC6 in a store-depletion insensitive manner, these TRPCs become sensitive to store depletion upon expression of an exogenous Orai. Thus, Orai-1, -2, and -3 enhanced thapsigargin-induced calcium entry by 50–150% in cells stably overexpressing either TRPC3 or TRPC6. Orai1 expression had no significant effect on endogenous, thapsigargin-induced calcium entry in wild-type cells (HEK-293, COS1), in HEK cells expressing a thapsigargin-sensitive variant of TRPC3 (TRPC3a), or in HEK cells overexpressing another membrane protein, V1aR. Single-channel cation currents present in membrane patches of TRPC3-overexpressing cells were suppressed by expression of Orai1. We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to these channels.

Potassium channel stimulation by natriuretic peptides through cGMP-dependent dephosphorylation

NATRIURETIC peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP)1,2, but the mechanism of cGMP action is unclear3. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP4,5; however, this mechanism is not involved in the action of cGMP on other channels6 or on Ca2+ channels in other cells7,8. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium9,10. In an electrophysiological study on rat pituitary tumour cells11, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems12.

Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels

Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Orai proteins. Another proposes SOCE channels to be composed of both Orai and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the Ca2+ stores that activates Ca2+ entry when stores are depleted. The role of Orai in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Orai proteins.

Calcium channel regulation by calcineurin, a Ca2+-activated phosphatase in mammalian brain

The enzymatic addition or removal of phosphate esters on serine and threonine hydroxyls alters the activity of many proteins that contribute to the characteristic structure and function of nerve cells. Recently, calcineurin, a major calmodulin-binding protein in mammalian brain, has been purified and identified as a Ca2+-activated protein phosphatase. Preliminary experiments suggest that calcineurin may limit Ca2+ influx through dihydropyridine-sensitive Ca2+ channels in the plasma membrane by dephosphorylating the channel, or a closely associated protein, and inactivating it.

Purification of a Fatty Acid-stimulated Protein-serine/threonine Phosphatase from Bovine Brain and Its Identification as a Homolog of Protein Phosphatase 5

An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with aM r of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 μm phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4–14-fold, with half-maximal stimulation at 50–100 μm, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator.

Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana

The receptor for activated C‐kinase 1 (RACK1) is a highly conserved WD40 repeat scaffold protein found in a wide range of eukaryotic species from Chlamydymonas to plants and humans. In tissues of higher mammals, RACK1 is ubiquitously expressed and has been implicated in diverse signaling pathways involving neuropathology, cellular stress, protein translation, and developmental processes. RACK1 has established itself as a scaffold protein through physical interaction with a myriad of signaling proteins ranging from kinases, phosphatases, ion channels, membrane receptors, G proteins, IP3 receptor, and with widely conserved structural proteins associated with the ribosome. In the plant Arabidopsis thaliana, RACK1A is implicated in diverse developmental and environmental stress pathways. Despite the functional conservation of RACK1‐mediated protein–protein interaction‐regulated signaling modes, the structural basis of such interactions is largely unknown. Here we present the first crystal structure of a RACK1 protein, RACK1 isoform A from Arabidobsis thaliana, at 2.4 Å resolution, as a C‐terminal fusion of the maltose binding protein. The structure implicates highly conserved surface residues that could play critical roles in protein–protein interactions and reveals the surface location of proposed post‐transcriptionally modified residues. The availability of this structure provides a structural basis for dissecting RACK1‐mediated cellular signaling mechanisms in both plants and animals.

Rac and Rho Mediate Opposing Hormonal Regulation of the Ether-A-Go-Go-Related Potassium Channel

BACKGROUND Previous studies of ion channel regulation by G proteins have focused on the larger, heterotrimeric GTPases, which are activated by heptahelical membrane receptors. In contrast, studies of the Rho family of smaller, monomeric, Ras-related GTPases, which are activated by cytoplasmic guanine nucleotide exchange factors, have focused on their role in cytoskeletal regulation. RESULTS Here we demonstrate novel functions for the Rho family GTPases Rac and Rho in the opposing hormonal regulation of voltage-activated, ether-a-go-go-related potassium channels (ERG) in a rat pituitary cell line, GH(4)C(1). The hypothalamic neuropeptide, thyrotropin-releasing hormone (TRH) inhibits ERG channel activity through a PKC-independent process that is blocked by RhoA(19N) and the Clostridium botulinum C3 toxin, which inhibit Rho signaling. The constitutively active, GTPase-deficient mutant of RhoA(63L) rapidly inhibits the channels when the protein is dialysed directly into the cell through the patch pipette, and inhibition persists when the protein is overexpressed. In contrast, GTPase-deficient Rac1(61L) stimulates ERG channel activity. The thyroid hormone triiodothyronine (T3), which antagonizes TRH action in the pituitary, also stimulates ERG channel activity through a rapid process that is blocked by Rac1(17N) and wortmannin but not by RhoA(19N). CONCLUSIONS Rho stimulation by G(13)-coupled receptors and Rac stimulation by nuclear hormones through PI3-kinase may be general mechanisms for regulating ion channel activity in many cell types. Disruption of these novel signaling cascades is predicted to contribute to several specific human neurological diseases, including epilepsy and deafness.

Stimulation of Kv1.3 potassium channels by death receptors during apoptosis in Jurkat T lymphocytes.

The loss of intracellular potassium is a pivotal step in the induction of apoptosis but the mechanisms underlying this response are poorly understood. Here we report caspase-dependent stimulation of potassium channels by the Fas receptor in a human Jurkat T cell line. Receptor activation with Fas ligand for 30 min increased the amplitude of voltage-activated potassium currents 2-fold on average. This produces a sustained outward current, ∼10 pA, at physiological membrane potentials during Fas ligand-induced apoptosis. Both basal and Fas ligand-induced currents were blocked completely by toxins that selectively inhibit Kv1.3 potassium channels. Kv1.3 stimulation required the expression of Fas-associated death domain protein and activation of caspase 8, but did not require activation of caspase 3 or protein synthesis. Furthermore, Kv1.3 stimulation by Fas ligand was prevented by chronic stimulation of protein kinase C with 20 nm phorbol 12-myristate 13-acetate during Fas ligand treatment, which also blocks apoptosis. Thus, Fas ligand increases Kv1.3 channel activity through the same canonical apoptotic signaling cascade that is required for potassium efflux, cell shrinkage, and apoptosis.

Activation of protein kinase C inhibits calcium-activated potassium channels in rat pituitary tumour cells.

1. The regulation of large‐conductance, calcium‐ and voltage‐dependent potassium (BK) channels by protein kinase C (PKC) was investigated in clonal rat anterior pituitary cells (GH4C1), which were voltage clamped at ‐40 mV in a physiological potassium gradient through amphotericin‐perforated patches. 2. Maximal activation of PKC by 100 nM phorbol 12, 13‐dibutyrate (PdBu) almost completely inhibited the voltage‐activated outward current through BK channels. In contrast PdBu had no significant effect on the residual outward current after block of BK channels with 2 mM TEA or 30 nM charybdotoxin. In single‐channel recordings from cell‐attached patches, PdBu reduced the open probability of BK channels more than eightfold with no significant effect on mean open lifetime or unitary conductance. 3. The effects of PdBu on BK channels were not mimicked by the 4 alpha‐isomer, which does not activate PKC, and were blocked almost completely by 25 microM chelerythrine, a specific, noncompetitive PKC inhibitor. 4. PdBu had no significant effect on the amplitude of the pharmacologically isolated, high voltage‐activated calcium current. 5. Inhibition of BK channel activity by PKC provides the first molecular mechanism linking hormonal activation of phospholipase C to sustained excitability in pituitary cells.

Somatostatin Stimulates BKCa Channels in Rat Pituitary Tumor Cells through Lipoxygenase Metabolites of Arachidonic Acid

The stimulation of large-conductance, calcium-activated (BK) potassium channels by somatostatin through protein dephosphorylation in rat pituitary tumor cells (White et al., Nature 351, 570-573, 1991) is blocked by drugs that interfere with arachidonic acid release by phospholipase A2 and metabolism by 5-lip-oxygenase. In contrast, higher concentrations of the same drugs had no effect on BK channel gating in cell-free patches, on the inhibition of adenylyl cyclase by somatostatin, or on the stimulation of BK channels by protein dephosphorylation through a cGMP-dependent pathway (White et al., Nature 361, 263-266, 1993). Exogenous arachidonic acid (1-20 muM) stimulated BK channel activity through protein dephosphorylation as effectively as somatostatin and was also blocked by inhibitors of lipoxygenases but not by inhibitors of phospholipase A2. These results support the hypothesis that lipoxygenase metabolites of arachidonic acid are second messengers linking pertussis toxin sensitive G-proteins to protein phosphatases regulating potassium channel activity (Armstrong and White, Trends Neurosci. 15, 403-408, 1992).