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

Stonehouse, Anthony H.; Pringle, J. H.; Norman, Robert I.; Stanfield, Peter; Conley, Edward C.; Brammar, William J.

doi:10.1007/s004180050429

Cited by 52 publications

(53 citation statements)

References 23 publications

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“…As an added complication, it is possible that K ir 2.2 exists as a heteromultimer with K ir 2.1 (25). Although K ir 2.3 may not contribute much to I K1 in the heart, its location in neurons (26,27) raises the possibility of a role in ischemia-induced excitotoxicity.…”

Section: Discussionmentioning

confidence: 99%

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Collins

Larson

2002

Journal of Biological Chemistry

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Inhibition of inward rectifier K؉ channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K ؉ channels K ir 2.1, K ir 2.2, and K ir 2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K ir 2.2 and K ir 2.3 currents but was without effect on K ir 2.1 currents. The rate of decline of current in FCCP was faster for K ir 2.3 than for K ir 2.2. K ir 2.3 was inhibited by 3 mM sodium azide (NaN 3 ), whereas K ir 2.1 and K ir 2.2 were not. K ir 2.2 was inhibited by 10 mM NaN 3 . All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K ir 2.3 was most sensitive to pH (pK ‫؍‬ 6.9), whereas K ir 2.1 was least sensitive (pK ‫؍‬ 5.9). For K ir 2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.

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Section: Discussionmentioning

confidence: 99%

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Collins

Larson

2002

Journal of Biological Chemistry

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“…17 Immunoprecipitates were subjected to SDS-PAGE 18 on 7 % polyacrylamide mini gels for 1.5 h at 120 V.…”

Section: Immunoprecipitationmentioning

confidence: 99%

“…17 HEK 293 cell transfection routinely. Cells were maintained in minimal essential medium with Earle's Salts supplemented with 10% foetal calf serum and 5% L-glutamine under antibiotic selection (SUR2A, 727 µg/ml G418; Kir6.2/SUR2A, 727 µg/ml G418, 364 µg/ml zeocin).…”

Section: Immunoprecipitationmentioning

confidence: 99%

“…Western blotting Twenty five microlitres of in vitro translated subunit protein reaction 17 or ventricular myocyte proteins (11 µg protein/ml) were denatured, separated by sodium dodecyl sulfate polyacrylamide (7.5%) gel electrophoresis (SDS-PAGE) 18 and transferred to nitrocellulose membranes (Hybond-C Super, Amersham Biosciences UK Limited, Little Chalfont, UK) with a wet-blot technique. 19 Membranes containing protein standards were stained with amido black stain (0.1 % amido black stain, 45 % methanol, 10 % acetic acid) and destained in 1 % acetic acid.…”

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Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes

Singh¹

2003

Journal of Molecular and Cellular Cardiology

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The subcellular distribution of K ATP channel subunits in rat isolated ventricular myocytes was investigated using a panel of subunit specific antisera. Kir6.1 subunits were associated predominantly with myofibril structures and were co-localized with the mitochondrial marker MitoTracker red (correlation coefficient (cc) = 0.63 ± 0.05 ).Anti-Kir6.1 antibodies specifically recognized a polypeptide of 48 kDa in mitochondrial membrane fractions consistent with the presence of Kir6.1 subunits in this organelle.Both Kir6.2 and SUR2A subunits were distributed universally over the sarcolemma.Lower intensity antibody associated immunofluorescence was detected intracellularly, which was correlated with the distribution of MitoTracker red in both cases (cc, Kir6.2, 0.56 ± 0.05; SUR2A, 0.61 ± 0.06). A polypeptide of 40 kDa was recognized by antiKir6.2 subunit antibodies in Western blots of both microsomal and mitochondrial membrane fractions consistent with the presence of this subunit in the sarcolemma and mitochondria. Similarly, SUR2A and SUR2B subunits were detected in Western blots of microsomal membrane proteins consistent with a sarcolemmal localization for these polypeptides. SUR2B subunits were shown in confocal microscopy to co-localize strongly with t-tubules (cc, 0.81 ± 0.05). Together the results indicate that Kir6.2 and SUR2A subunits predominate in the sarcolemma of ventricular myocytes consistent with a Kir6.2/SUR2A subunit combination in the sarcolemmal K ATP (sarcK ATP ) channel.Kir6.1, Kir6.2 and SUR2A subunits were demonstrated in mitochondria. Combinations of these subunits would not explain the reported pharmacology of the mitochondrial K ATP (mitoK ATP ) channel (Lui et al. 2001. Mol. Pharmacol. 59:225-230) suggesting the possibility of further unidentified components of this channel.3

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“…Furthermore, Kir2.3 binds to PSD-95, which scaffolds various functional proteins at the postsynaptic membrane of asymmetric synaptic junctions (5,7,23,30). Although these results suggest that Kir2.3 is localized at the postsynaptic membrane of excitatory synapses, light microscopy indicated that neuronal Kir2.3 distributes diffusely in the nuclei and on the plasma membrane of pyramidal cells in the CA3 region of rat hippocampus (33). To resolve this paradox, we produced a polyclonal antibody specific to Kir2.3 and examined the localization of the channel protein in mouse brain at light-and electron-microscopic levels.…”

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confidence: 88%

Inward rectifier K⁺ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

Inanobe

Fujita

Ito

et al. 2002

American Journal of Physiology-Cell Physiology

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Classical inwardly rectifying K+ channels (Kir2.0) are responsible for maintaining the resting membrane potential near the K+ equilibrium potential in various cells, including neurons. Although Kir2.3 is known to be expressed abundantly in the forebrain, its precise localization has not been identified. Using an antibody specific to Kir2.3, we examined the subcellular localization of Kir2.3 in mouse brain. Kir2.3 immunoreactivity was detected in a granular pattern in restricted areas of the brain, including the olfactory bulb (OB). Immunoelectron microscopy of the OB revealed that Kir2.3 immunoreactivity was specifically clustered on the postsynaptic membrane of asymmetric synapses between granule cells and mitral/tufted cells. The immunoprecipitants for Kir2.3 obtained from brain contained PSD-95 and chapsyn-110, PDZ domain-containing anchoring proteins. In vitro binding assay further revealed that the COOH-terminal end of Kir2.3 is responsible for the association with these anchoring proteins. Therefore, the Kir channel may be involved in formation of the resting membrane potential of the spines and, thus, would affect the response of N-methyl-d-aspartic acid receptor channels at the excitatory postsynaptic membrane.

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

Cited by 52 publications

References 23 publications

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes

Inward rectifier K⁺ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

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

Cited by 52 publications

References 23 publications

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes

Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

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Inward rectifier K⁺ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses