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Biomedical subjects

D E Clapham

Publications and source records attributed to D E Clapham.

At least 55 records · Page 3Linked to original sources
PubMed1998

Evidence for direct physical association between a K+ channel (Kir6.2) and an ATP-binding cassette protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K+ channel.

Structurally unique among ion channels, ATP-sensitive K+ (KATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K+ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2deltaC37). Kir6.2deltaC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2deltaC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 exhibited single-channel activity characteristic of pancreatic KATP channels. Kir6.2deltaC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K+ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a KATP channel.

ATP-Binding Cassette Transporters↗
PubMed1997

Partial structure, chromosome localization, and expression of the mouse Icln gene.

The molecular identities of most volume-regulated ion channel proteins and putative regulatory elements are currently unknown. Recently, a role for a nucleotide-sensitive chloride conductance regulator, ICln, in the function of a ubiquitous volume-regulated chloride channel has been suggested. Here, we report the cloning of a fragment of the mouse Icln gene and identification of probable Icln pseudogenes. The functional Icln gene was mapped independently to human chromosome 11q13.5-q14 and mouse chromosome 7 (50.3 cM). ICln mRNA was shown to be abundantly expressed and evenly distributed in all mouse tissues examined and at four stages of embryonic development, consistent with the proposed role of ICln in the regulation of a ubiquitous chloride channel.

Amino Acid Sequence↗
PubMed1997

Partial structure, chromosome localization, and expression of the mouse Girk4 gene.

The G protein-gated potassium channel IKACh constitutes part of a signaling pathway that mediates the negative chronotropic and inotropic effects of acetylcholine on cardiac physiology. Similar or identical ion channels regulate the excitability of many neurons in response to neurotransmitters. IKACh is composed of two homologous subunits, GIRK1 and GIRK4. Here we describe a partial genomic structure of the mouse Girk4 gene. Two exons containing the complete protein-coding sequence were identified. Girk4 was mapped to mouse chromosome 9 (13 cM), consistent with the mapping of human GIRK4 to chromosome 11q23-ter. GIRK4 mRNA was found mainly in mouse heart, with trace levels detected in brain, kidney, lung, and spleen. No detectable levels were observed in skeletal muscle, liver, and testis. The onset of GIRK4 mRNA expression in the developing mouse occurs between Embryonic Days 7 and 11, consistent with the appearance and function of the mouse heart.

Animals↗
PubMed1997

Nuclear calcium and the regulation of the nuclear pore complex.

In eukaryotic cells the nucleus and its contents are separated from the cytoplasm by the nuclear envelope. Macromolecules, as well as smaller molecules and ions, can cross the nuclear envelope through the nuclear pore complex. Molecules greater than approx. 60 kDa and containing a nuclear localization signal are actively transported across the nuclear membranes, but there has been little evidence for regulatory mechanisms for smaller molecules and ions. Recently, diffusion across the nuclear envelope has been observed to be regulated by nuclear cisternal Ca2+ concentrations. Following depletion of Ca2+ from the nuclear store by inositol 1,4,5-trisphosphate or Ca2+ chelators, a fluorescent 10 kDa marker molecule was no longer able to enter the nucleus. Distinct conformational states of the nuclear pore complexes depended on the Ca2+ filling state of the nuclear envelope, supporting the assumption that a switch in the conformation of the nuclear pore complex may control the transport of intermediate-sized molecules across the nuclear envelope. Thus nuclear Ca2+ stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. The physiological significance of this finding is currently unknown.

Animals↗
PubMed1997

Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3.

The fluorescent indicator Fluo-3 is widely used to monitor the calcium concentration ([Ca2+]) in the cytoplasm and nucleus of various cells. Estimates of nuclear [Ca2+] are based on the assumption of identical behavior of Fluo-3 in different cellular compartments. The assumption is not valid if the fluorescence properties of the dye are altered by the nuclear environment, independent of the [Ca2+]. To determine the effects of the nucleoplasm on the behavior of Fluo-3, we applied laser scanning confocal microscopy and spectrophotometry to measure fluorescence intensity as well as emission and absorbance spectra of the Ca2+ indicator, Fluo-3. Spectra were measured in intact Xenopus oocytes, neuroblastoma cells, and cytoplasmic and nucleoplasmic homogenates. The fluorescence signal in intact cells loaded with Fluo-3 was approximately 2-times higher in the nucleus when compared to the cytoplasm. The fluorescence intensity of Fluo-3 in nucleoplasmic homogenates was higher than in cytoplasmic homogenates or internal buffers even when [Ca2+] was clamped. Despite identical [Ca2+], pH, and temperature, the emission and absorbance spectra of Fluo-3 from nuclear homogenates displayed a higher fluorescence at each wavelength measured when compared to spectra from cytoplasmic homogenates or internal buffer solutions, and saturated above 100 nM. These findings demonstrate that the composition of the nucleoplasm changes the fluorescence properties of the calcium indicator Fluo-3. Consequently, analysis of nuclear calcium dynamics must take into account the distinct behavior of Fluo-3 in different cellular compartments.

Aniline Compounds↗
PubMed1997

G protein beta gamma subunits.

Guanine nucleotide binding (G) proteins relay extracellular signals encoded in light, small molecules, peptides, and proteins to activate or inhibit intracellular enzymes and ion channels. The larger G proteins, made up of G alpha beta gamma heterotrimers, dissociate into G alpha and G beta gamma subunits that separately activate intracellular effector molecules. Only recently has the G beta gamma subunit been recognized as a signal transduction molecule in its own right; G beta gamma is now known to directly regulate as many different protein targets as the G alpha subunit. Recent X-ray crystallography of G alpha, G beta gamma, and G alpha beta gamma subunits will guide the investigation of structure-function relationships.

Binding Sites↗
PubMed1997

The molecular control of cardiac ion channels.

The heart is composed of specialized cells ranging from sinoatrial nodal pacing cells with relatively few contractile elements to paced ventricular cells packed with contractile muscle protein. Arising in the sinoatrial node and transmitted throughout the atria, atrioventricular (AV) node, His-Purkinje system, and the ventricles, the cardiac action potential precisely activates distinct populations of ion channels. Most, if not all, ion channels are modulated by signal transduction mechanisms which shape the action potential. Six principal ion currents contribute to the nodal and pacemaking cell action potential and ten underlie the myocardial action potential. Other ion channels are specially designed for modulation of the action potential shape.

Acetylcholine↗
PubMed1996

Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ stores.

The nuclear pore complex (NPC) is essential for the transit of molecules between the cytoplasm and nucleoplasm of a cell and until recently was thought to allow intermediate-sized molecules (relative molecular mass of approximately 10,000) to diffuse freely across the nuclear envelope. However, the depletion of calcium from the nuclear envelope of Xenopus laevis oocytes was shown to regulate the passage of intermediate-sized molecules. Two distinct conformational states of the NPC were observed by field emission scanning electron microscopy and atomic force microscopy. A central plug occluded the NPC channel after nuclear calcium stores had been depleted and free diffusion of intermediate-sized molecules had been blocked. Thus, the NPC conformation appears to gate molecular movement across the nuclear envelope.

Animals↗
PubMed1996

Intracellular signalling: more jobs for G beta gamma.

G-protein-coupled receptors that modulate Ca2+ channels that mediate synaptic transmission seem to use the G beta gamma, and not the G alpha, subunits of the trimeric G protein to slow and inhibit the Ca2+ currents.

Calcium Channels↗
PubMed1996

Nonselective and G betagamma-insensitive weaver K+ channels.

Homozygous weaver mice are profoundly ataxic because of the loss of granule cell neurons during cerebellar development. This granule cell loss appears to be caused by a genetic defect in the pore region (Gly156-->Ser) of the heterotrimeric guanine nucleotide-binding protein (G protein)-gated inwardly rectifying potassium (K+) channel subunit (GIRK2). A related subunit, GIRK1, associates with GIRK2 to constitute a neuronal G protein-gated inward rectifier K+ channel. The weaver allele of the GIRK2 subunit (wvGIRK2) caused loss of K+ selectivity when expressed either as wvGIRK2 homomultimers or as GIRK1-wvGIRK2 heteromultimers. The mutation also let to loss of sensitivity to G protein betagamma dimers. Expression of wvGIRK2 subunits let to increased cell death, presumably as a result of basal nonselective channel opening.

Amino Acid Sequence↗
PubMed1996

The K+ channel inward rectifier subunits form a channel similar to neuronal G protein-gated K+ channel.

G protein-activated inwardly rectifying K+ channel subunits GIRK1 (Kir 3.1), GIRK2 (Kir 3.2), and CIR (Kir 3.4) were expressed individually or in combination in Xenopus oocytes and CHO cells. GIRK1 coexpressed with CIR or GIRK2, produced currents up to 10-fold larger than any of the subunits expressed alone. No such clear synergistic effects were observed upon coexpression of CIR/GIRK2 under the same conditions. Coexpression of G protein beta gamma (G beta 1 gamma 2) increased the current through GIRK1/GIRK2 and GIRK2 channels. G beta gamma subunits purified from bovine brain, increased channel activity 50-1000-fold in patches from cells expressing GIRK1/GIRK2 or GIRK2 alone. The single GIRK1/GIRK2 channels resembled previously described neuronal G protein-gated K+ channels. In contrast, single GIRK2 channels were short-lived and unlike any previously described neuronal K+ channel. We propose that some neuronal G protein-activated inward rectifier K+ channels may be formed by a GIRK1/GIRK2 heteromultimer and that G beta gamma activation may involve both subunits.

Animals↗
PubMed1996

Localization and interaction of epitope-tagged GIRK1 and CIR inward rectifier K+ channel subunits.

GIRK1 and CIR are G-protein activated inward rectifier K+ channel subunits that combine to form the heteromultimer IKACh, the G beta gamma-activated atrial K channel responsible for the vagal slowing of heart rate. Epitope-tagged channel subunits were constructed by the introduction of distinct six amino acid epitopes into the C-termini or putative extracellular domains of GIRK1 and CIR. Carboxyl-terminal tagged subunits were activated by purified G beta gamma subunits in inside-out patches when expressed in Cos cells. Interestingly, insertion of three amino acids into the putative extracellular domain of GIRK1 resulted in an inactive subunit that acted as a dominant negative subunit when coexpressed with wild type GIRK1 and CIR in Xenopus oocytes. The epitope-tagged CIR-AU1 subunit coimmunoprecipitated GIRK1-AU5 from metabolically labeled Cos cells. Immunofluorescence labeling of Cos cells localized GIRK1-AU5 to internal cytoskeletal structures that co-stained with antibodies against the intermediate filament protein, vimentin. CIR-AU1 localized primarily to the plasma membrane. Double immunofluorescence labeling showed that GIRK1-AU5 plasma membrane staining was detectable only when coexpressed with CIR-AU1.

Animals↗
PubMed1996

Inward rectification of the IRK1 K+ channel reconstituted in lipid bilayers.

Inwardly rectifying potassium (K+) channels (IRK1) were incorporated into lipid bilayers to test the relative contributions of various mechanisms to inward rectification. IRK1 channels were expressed in Xenopus laevis oocytes and oocyte membrane vesicles containing the channels were fused with lipid bilayers. The major properties of the IRK1 channel were similar whether measured in the oocyte membrane or lipid bilayer; the single channel conductance was 21 pS in 140 mM symmetrical [K+] and varied as a square root of external [K+]. Importantly, IRK1 channels display voltage-dependent inward rectification in the absence of divalent ions or charged regulators such as spermine, indicating that they possess an intrinsic rectification mechanism. Although rectification was significantly increased by either Mg2+ or spermine added to the cytoplasmic face of the channel, their effects could not be explained by simple block of the open pore. The Hille and Schwartz (1978) model, originally proposed to explain inward rectification by singly charged blocking particles, cannot be used to explain rectification by multiply charged blocking particles. As an alternative, we propose that in addition to a slow gating mechanism producing long lasting open and closed states, there is a distinct, intrinsic fast gating process amplified by cytoplasmic Mg2+ and/or polyamine binding to the channel.

Animals↗
PubMed1996

Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes.

Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels.

Acetylcholine↗