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 Ca(2+), 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 Ca(2+)-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 Ca(2+)-activated K(+) and Cl(-) currents may be involved in cell activation.

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-anchoring proteins (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 glutathione S-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.

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.

The possible role of a disulphide bond in forming functional Kir2.1 potassium channels.

The role of two cysteine residues--Cys122 and Cys154--in the structure of the strong inward rectifier K+ channel, Kir2.1, has been investigated using site-directed mutagenesis and electrophysiology. Such cysteine residues are conserved across the inward rectifier family and may be expected to form a crucial disulphide bond. Our experiments show that when the cysteines are absent, the protein is expressed, but the channels are not functional, suggesting that the disulphide bond is essential for correct channel assembly. However, reducing agents applied extracellularly have little effect on current amplitude in wild-type, so that, once the channel is assembled correctly in the membrane, the disulphide bonds are no longer essential for function. Molecular modelling suggests that a disulphide bond is formed--this may be either an intra- or an inter-subunit.

The dependence of Ag+ block of a potassium channel, murine kir2.1, on a cysteine residue in the selectivity filter.

Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.

The selectivity filter of a potassium channel, murine kir2.1, investigated using scanning cysteine mutagenesis.

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. 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. 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. 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. 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-pi interactions and by backbone carbonyl groups (Thr142 and Gly144).

Characterization of a ubiquitinated protein which is externally located in African swine fever virions.

An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions. The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence of E. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein.

A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A.T.C.C. 6633.

A variant of the peptide antibiotic subtilin has been isolated from Bacillus subtilis A.T.C.C. 6633, and its structure has been shown to be [N alpha-succinyl-Trp1]subtilin. The chemical structure of a fragment derived by tryptic hydrolysis of the variant is shown to be N alpha-succinyl-Trp-Lys by 1H and 13C n.m.r., fast-atom-bombardment m.s. and total chemical synthesis [N alpha-Succinyl-Trp1]-subtilin is produced later in the growth of the bacterium than is subtilin; reverse-phase h.p.l.c. analysis shows that after 24 h growth the ratio subtilin/[N alpha-succinyl-Trp1]subtilin is approx. 1:2. Although [N alpha-succinyl-Trp1]subtilin retains significant antibacterial activity, it is 10-20 times less active than subtilin.

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 lanthionine and 3-methyllanthionine residues. These results suggest that in aqueous solution subtilin and the homologous peptide nisin have similar conformations.

Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii.

An isocitrate dehydrogenase able to function with either NADP or NAD as coenzyme was purified to homogeneity from cell-free extracts of the purple photosynthetic eubacterium Rhodomicrobium vannielii using a rapid two-step procedure involving dye-ligand affinity chromatography. The enzyme was obtained in 60% yield with specific activities of 23 U.mg protein-1 (NADP-linked reaction) and 18.5 U.mg protein-1 (NAD-linked reaction). The purified enzyme was monomeric and migrated with an approximate Mr of 75,000-80,000 on both SDS/PAGE and non-denaturing PAGE. Affinity constants (Km values) of 2.5 microM for NADP and 0.77 mM for NAD and values for kcat/Km of 981,200 min-1.mM-1 (NADP) and 2455 min-1.mM-1 (NAD) indicated a greater specificity for NADP compared to NAD. A number of metabolites were examined for possible differential regulatory effects on the NADP- and NAD-linked reactions, using a dual-wavelength assay. Oxaloacetate was found to be an effective inhibitor of both reactions and the enzyme was also sensitive to concerted inhibition by glyoxylate and oxaloacetate. The amino-acid composition and the identity of 39 residues at the N-terminus were determined and compared to other isocitrate dehydrogenases. The results suggested a relationship between the Rm. vannielii enzyme and the monomeric isocitrate dehydrogenase isoenzyme II from Vibrio ABE-1.