Mark L. Leyland

The PSD95–nNOS interface: a target for inhibition of excitotoxic p38 stress-activated protein kinase activation and cell death

The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream effectors of excitotoxic cell death. Although the postsynaptic density protein PSD95 can recruit the calcium-dependent neuronal NO synthase (nNOS) to the mouth of the calcium-permeable NMDA receptor, and depletion of PSD95 inhibits excitotoxicity, the possibility that selective uncoupling of nNOS from PSD95 might be neuroprotective is unexplored. The relationship between excitotoxic stress–generated NO and activation of p38, and the significance of the PSD95–nNOS interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death. Experiments using a panel of decoy constructs targeting the PSD95–nNOS interaction suggest that this interaction and subsequent NO production are critical for glutamate-induced p38 activation and the ensuing cell death, and demonstrate that the PSD95–nNOS interface provides a genuine possibility for design of neuroprotective drugs with increased selectivity.

Structure‐activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin

The post‐translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth‐inhibition activity. Removal of the C‐terminal five residues leads to approximately a 10‐fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100‐fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin1−12 is inactive itself, and specifically antagonises the growth‐inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.

Residues beyond the selectivity filter of the K+ channel Kir2.1 regulate permeation and block by external Rb+ and Cs+

1 Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage‐dependent manner, characteristic of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lowered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. 2 A model of the Kir2.1 channel, built by homology with the structure of the Streptomyces lividans K+ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore‐forming P‐region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage. 3 An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore‐lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non‐polar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. 4 We tested the hypothesis that Asp172 – a residue implicated in channel gating by polyamines – formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb+ and Cs+ currents were measured in this mutant. 5 We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild‐type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.

Targeting of an A Kinase-anchoring Protein, AKAP79, to an Inwardly Rectifying Potassium Channel, Kir2.1

Protein kinase A (PKA) is targeted to discrete subcellular locations close to its intended substrates through interaction with A kinase-anchoringproteins (AKAPs). Ion channels represent a diverse and important group of kinase substrates, and it has been shown that membrane targeting of PKA through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of several classes of ion channel. Here, we investigate the effect of AKAP79, a membrane-associated multivalent-anchoring protein, upon the function and modulation of the strong inwardly rectifying potassium channel, Kir2.1. Functionally, the presence of AKAP79 enhanced the response of Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a pool of PKA anchored close to the channel. Antibodies directed against a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79, indicating that the two proteins exist together in a complex within intact cells. In support of this, glutathione S-transferase fusion proteins of both the intracellular N and C domains of Kir2.1 isolated AKAP79 from cell lysates, while glutathioneS-transferase alone failed to interact with AKAP79. Together, these findings suggest that AKAP79 associates directly with the Kir2.1 ion channel and may serve to anchor kinase enzymes in close proximity to key channel phosphorylation sites.

CRACM/Orai ion channel expression and function in human lung mast cells

Background Influx of extracellular Ca2+ into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca2+ influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca2+ release–activated Ca2+ current are candidates. Objectives To investigate the expression and function of CRACM channels in HLMCs. Methods CRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus. Results CRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca2+-selective current obtained under both conditions was blocked by 10 μM La3+ and Gd3+, known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers—GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca2+ influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C4, and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue. Conclusions The presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca2+ influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases.

Voltage‐dependent and calcium‐activated ion channels in the human mast cell line HMC‐1

The mechanisms underlying the recruitment, differentiation, and sustained activation of mast cells in disease are likely to include modulation of ion channels. Specific Ca2+, K+, and Cl− conductances have been identified in rodent mast cells, but there are no equivalent data on human mast cells. We have used the whole‐cell patch‐clamp technique to characterize macroscopic ion currents in both the human mast cell line HMC‐1 and human skin mast cells (HSMCs) at rest and in HMC‐1 after activation with calcium ionophore. HSMCs were electrically silent at rest. In contrast, HMC‐1 expressed a strong outwardly rectifying voltage‐dependent Cl− conductance characteristic of ClC‐4 or ClC‐5 and a small inwardly rectifying K+ current not carried by the classical Kir family of K+ channels. Calcium ionophore induced the appearance of outwardly rectifying Ca2+‐activated Cl− and K+ currents, while hypotonicity induced another outwardly rectifying conductance typical of ClC‐3. Reverse transcription‐PCRs confirmed that mRNAs for the voltage‐dependent Cl− channels ClC‐3 and –5 were expressed. This is the first definitive description of a ClC‐4/5‐like current in a native leukocyte. We suggest that this current may contribute to the malignant phenotype while the Ca2+‐activated K+ and Cl− currents may be involved in cell activation.

An Alternatively Spliced Isoform of PSD-93/Chapsyn 110 Binds to the Inwardly Rectifying Potassium Channel, Kir2.1

Inwardly rectifying potassium (Kir) channels are prime determinants of resting membrane potential in neurons. Their subcellular distribution and surface density thus help shape neuronal excitability, yet mechanisms governing the membrane targeting and localization of Kir channels are poorly understood. Here we report a direct interaction between the strong inward rectifier, Kir2.1, and a recently identified splice variant of postsynaptic density-93 (PSD-93), a protein involved the subcellular targeting of ion channels and glutamate receptors at excitatory synapses. Yeast two-hybrid screening of a human brain cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding the first two PDZ domains of PSD-93, but with an extended N-terminal region that diverged from other PSD-93 isoforms. This clone represented the human homologue of the mouse PSD-93 splice variant, PSD-93δ. Reverse transcription-polymerase chain reaction analysis showed diffuse low level PSD-93δ expression throughout the brain, with significantly higher levels in spinal cord. In vitro binding studies revealed that a type I PDZ recognition motif at the extreme C terminus of the Kir2.1 mediates interaction with all three PDZ domains of PSD-93δ, and association between Kir2 channels and PSD-93δ was confirmed further by the ability of anti-Kir2.1 antibodies to coimmunoprecipitate PSD-93δ from rat spinal cord lysates. Functionally, coexpression of Kir2.1 and PSD-93δ had no discernible effect upon channel kinetics but resulted in cell surface Kir2.1 clustering and suppression of channel internalization. We conclude that PSD-93δ is potentially an important regulator of the spatial and temporal distribution of Kir2 channels within neuronal membranes of the central nervous system.

The selectivity filter of a potassium channel, murine Kir2.1, investigated using scanning cysteine mutagenesis

1 We have produced a structural model of the pore‐forming H5 (or P) region of the strong inward rectifier K+ channel, Kir2.1, based initially on an existing molecular model of the pore region of the voltage‐gated K+ channel, Kv1.3. 2 Cysteine‐scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residues in H5 whose side chains line the ion conduction pathway. 3 Mutations made in eight positions within the highly conserved H5 region resulted in apparently non‐functional channels. Constructing covalently linked dimers, which carry a cysteine substitution in only one of the linked subunits, rescued six of these mutants; a covalently linked tetramer, carrying a cysteine substitution on only one of the linked subunits, rescued a further mutant. 4 Our results using the dimers and tetramers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100‐200 nM) and therefore that their side chains line the channel pore. 5 We conclude that the topology of the Kir pore is similar, but not identical, to that of Kv channels. Additionally, the molecular model suggests that selectivity may be conferred both by aromatic residues (Tyr145 and Phe147) via cation‐π interactions and by backbone carbonyl groups (Thr142 and Gly144).

Sequence-specific resonance assignment and conformational analysis of subtilin by 2D NMR

Subtilin, a 32‐amino acid peptide with potent antimicrobial activity, has been isolated from Bacillus subtilis ATCC6633. The chemical structure has been confirmed by the unambiguous sequence‐specific assignment of its 1H NMR spectrum. Detailed NMR analysis revealed that subtilin is a rather flexible molecule; the only observed conformational contraints were those imposed by the cyclic structures created by the tanthionine and 3‐methyllanthionine residues. These results suggest that in aqueous solution subtilin and the homologous peptide nisin have similar conformation.

A novel FcεRIβ-chain truncation regulates human mast cell proliferation and survival

Mast cells contribute to allergy through IgE‐dependent activation via the high‐affinity IgE receptor FcεRI. The role of the FcεRIβ chain (MS4A2) in mast cell function is not understood fully, although it serves to amplify FcεRI‐dependent signaling. We demonstrate the expression of a novel MS4A2 truncation lacking exon 3 in human mast cells termed MS4A2trunc. MS4A2trunc gene expression was regulated negatively by the mast cell growth factor stem cell factor (SCF), and its expression was not detected in the SCF receptor gain‐of‐function human mast cell line HMC‐1. Unlike MS4A2, MS4A2trunc did not traffic to the cytoplasmic membrane but instead was associated with the nuclear membrane. Overexpression of MS4A2trunc induced human lung mast cell death and profoundly inhibited HMC‐1 cell proliferation by inducing G2‐phase cell cycle arrest and apoptosis. Thus, we have identified a novel splice variant of MS4A2 that might be important in the regulation of human mast cell proliferation and survival. This finding demonstrates that the MS4A2 gene has multiple roles, extending beyond the regulation of acute allergic responses. By understanding the mechanisms regulating its function, it might be possible to induce its expression in mast cells in vivo, which could lead to better treatments for diseases such as mastocytosis and asthma.—Cruse, G., Kaur, D., Leyland, M., Bradding, P. A novel FcεRIβ‐chain truncation regulates human mast cell proliferation and survival. FASEB J. 24, 4047–4057 (2010). www.fasebj.org