Edward C. Conley

A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane‐spanning alpha‐helix of the murine inward rectifier, IRK1, by site‐directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.

The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole‐cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e‐fold as the driving force on K+(V‐EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current‐voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.

Resting and Activation-Dependent Ion Channels in Human Mast Cells

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca2+, K+, and Cl− conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca2+-activated K+ current exhibiting weak inward rectification, which polarized the cells to around −40 mV and a smaller outwardly rectifying Ca2+-independent Cl− conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca2+-activated K+ currents and Ca2+-independent Cl− currents developing in ∼50% of cells. In both cell types, the K+ current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca2+-activated K+ channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C4 release, indicating that the Ca2+-activated K+ current may enhance, but is not essential for, the release of these mediators.

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies.

Abstract Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

Elevated extracellular [K] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release

Efflux of intracellular K(+) and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K(+)] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K(+) ion surrogate (86)Rb(+), we have demonstrated an efflux of intracellular K(+) during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K(+) ions in apoptosis, cytoprotection by elevated extracellular [K(+)] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K(+)] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing approximately 700 kDa complex and an inactive approximately 1.4 MDa complex. Increasing [K(+)] inhibited caspase activation by preventing formation of the approximately 700 kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K(+)] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress.

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.

The dependence of Ag+ block of a potassium channel, murine Kir2.1, on a cysteine residue in the selectivity filter

1 Externally applied Ag+ (100‐200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. 2 Mutation to serine of a cysteine residue at position 149 in the pore‐forming H5 region of Kir2.1 abolished Ag+ blockage. 3 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. 4 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. 5 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.

Co‐localization of the Inwardly Rectifying Potassium Ion Channel, Kir2.2, and the Substance P Receptor in Single Locus Coeruleus Neurons

Inwardly rectifying potassium (Kir) channels are expressed in a wide range of excitable and non-excitable cells, where they set the resting membrane potential. Because Kirs are open in the range of the resting potential, their modulation is an important factor in the regulation of cellular excitability. In the noradrenergic neurons of locus coeruleus (LC), the neuropeptide substance P (SP) has been shown to suppress Kir activity.1,2 In this report we demonstrate, by immunohistochemistry with site-directed polyclonal antibodies, the co-localization of Kir2.2 and substance P receptor (SPR) proteins in single LC neurons and associated oligodendroglia. In addition, we show a nuclear localization in LC neurons for Kir2.2 protein. This was supported by a nuclear signal with Kir2.2 antibodies obtained in Chinese hamster ovary (CHO) cells transiently transfected with Kir2.2. Expression of Kir2.2 protein could not be detected in untransfected cells. The Kir2.2 antibody detected a single 62 kD band on Western blots containing rat brain nuclear and plasma membrane protein fractions. This band was blocked by incubation with the 10 μg/ml Kir2.2 antigenic peptide (data not shown). Similarly Kir2.2 immunostaining on rat brain tissue sections was also blocked by incubation with the Kir2.2 antigenic peptide (data not shown). Adjacent coronal cerebellar rat brain tissue sections (10 μm deep, paraffin-embedded) were immunostained with the affinity purified SPR antibody (1:500), or the affinity purified Kir2.2 antibody (12 μg/ml). The large diameter (∼40 μm) of LC neurons made it possible to identify cells present in serial tissue sections. Strong nuclear and plasma membrane signals in neurons were observed for Kir2.2, with a clear nuclear signal also in oligodendroglia (FIG. 1A). SPR was strongly expressed on the plasma membranes of neurons and oligodendroglia, as well as neural processes within the LC (FIG. 1B). In order to establish co-expression of Kir2.2 and SPR, the Macromedia XRES program was used to overlay the two adjacent tissue sections. This overlay demonstrated a co-localization of SPR and Kir2.2 proteins in single neurons and oligodendroglia of the LC (FIG. 1C). This is consistent with previous