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D E Clapham

Publications and source records attributed to D E Clapham.

At least 73 records · Page 4Linked to original sources
PubMed1996

Nuclear ion channels and regulation of the nuclear pore.

Multiple K+, Cl-, and nonselective cationic nuclear channels have been identified in a variety of cell types from coconut endosperm to cardiac myocytes. To date, only one ligand-gated nuclear channel has been characterized-the InsP3R-but it is likely that others are also present. The InsP3R channel has been implicated in two important nuclear events: (1) nuclear vesicle fusion (Sullivan et al., 1993) and (2) regulation of molecular transport through the nuclear pore (Stehno-Bittel et al., 1993) and (2) 1995b). Regulation of passive diffusion across the nuclear envelope by the nuclear Ca2+ store may play an important role in nuclear events ranging from apoptosis to cell division.

Animals↗
PubMed1995

Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs.

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.

Animals↗
PubMed1995

The G protein beta gamma subunit transduces the muscarinic receptor signal for Ca2+ release in Xenopus oocytes.

At least 30 G protein-linked receptors stimulate phosphatidylinositol 4,5-bisphosphate phosphodiesterase (phospholipase C beta, PLC beta) through G protein subunits to release intracellular calcium from the endoplasmic reticulum (Clapham, D. E. (1995) Cell 80, 259-268). Although both G alpha and G beta gamma G protein subunits have been shown to activate purified PLC beta in vitro, G alpha q has been presumed to mediate the pertussis toxin-insensitive response in vivo. In this study, we show that G beta gamma plays a dominant role in muscarinic-mediated activation of PLC beta by employing the Xenopus oocyte expression system. Antisense nucleotides and antibodies to G alpha q/11 blocked the m3-mediated signal transduction by inhibiting interaction of the muscarinic receptor with the G protein. Agents that specifically bound free G beta gamma subunits (G alpha-GDP and a beta-adrenergic receptor kinase fragment) inhibited acetylcholine-induced signal transduction to PLC beta, and injection of G beta gamma subunits into oocytes directly induced release of intracellular Ca2+. We conclude that receptor coupling specificity of the G alpha q/G beta gamma heterotrimer is determined by G alpha q; G beta gamma is the predominant signaling molecule activating oocyte PLC beta.

Acetylcholine↗
PubMed1995

Diffusion across the nuclear envelope inhibited by depletion of the nuclear Ca2+ store.

Intact, isolated nuclei and a nuclear membrane (ghost) preparation were used to study regulation of the movement of small molecules across the Xenopus laevis oocyte nuclear membrane. In contrast to models of the nuclear pore complex, which assume passive bidirectional diffusion of molecules less than 70 kilodaltons, diffusion of intermediate-sized molecules was regulated by the nuclear envelope calcium stores. After depletion of nuclear store calcium by inositol 1,4,5-trisphosphate or calcium chelators, fluorescent molecules conjugated to 10-kilodalton dextran were unable to enter the nucleus. Dye exclusion after calcium store depletion was not dependent on the nuclear matrix because it occurred in nuclear ghosts lacking nucleoplasm. Smaller molecules and ions (500-dalton Lucifer yellow and manganese) diffused freely into the core of the nuclear ghosts and intact nuclei even after calcium store depletion. Thus, depletion of the nuclear calcium store blocks diffusion of intermediate-sized molecules.

Adenosine Triphosphate↗
PubMed1995

G beta gamma binds directly to the G protein-gated K+ channel, IKACh.

The cardiac G protein-gated K+ channel, IKACh, is activated by application of purified and recombinant beta and gamma subunits (G beta gamma) of heterotrimeric G proteins to excised inside-out patches from atrial membranes (Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D. E. (1987) Nature 325, 321-326; Wickman, K., Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B., Linder, M. E., Gilman, A., and Clapham, D.E. (1994) Nature 368, 255-257). Cardiac IKACh is composed of two inward rectifier K+ channel subunits, GIRK1 and CIR (Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We show that G beta gamma directly binds to immunoprecipitated cardiac IKACh as well as to recombinant CIR and GIRK1 subunits, with dissociation constants (Kd) of 55, 50, and 125 nM, respectively. In each case, binding appeared specific as judged by competition of unlabeled G beta gamma with radiolabeled G beta gamma and inhibition of binding by antigenic peptide or G alpha-GDP, but not G alpha-GTP gamma S (guanosine 5'-3-O-(thio)triphosphate). In contrast, G alpha (GTP gamma S- or GDP-bound) did not bind to the native channel. We conclude that G beta gamma binds directly and specifically to IKACh via interactions with both CIR and GIRK1 subunits to gate the channel.

Animals↗
PubMed1995

The cardiac inward rectifier K+ channel subunit, CIR, does not comprise the ATP-sensitive K+ channel, IKATP.

Cardiac IKACh is comprised of two inwardly rectifying K+ channel subunits, CIR and GIRK1 (Krapivinsky, G., Gordon, E. G., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). A cardiac protein virtually identical to CIR, termed rcKATP-1 (Ashford, M. L. J., Bond, C. T., Blair, T. A., and Adelman, J. P. (1994) Nature 370, 456-459), was reported to form an ATP-sensitive inwardly rectifying K+ channel, IKATP. We attempted to determine whether CIR alone or together with an unknown protein(s) participated in the formation of cardiac IKATP. Expression of CIR in insect, oocyte, and mammalian cell systems did not increase the appearance of ATP-sensitive currents, but rather gave rise to unique strongly inwardly rectifying, G protein-regulated K+ currents. CIR protein is found exclusively in atria, in contrast to the predominance of IKATP functional activity in ventricle. Also, CIR was completely depleted from heart membrane after immunodepletion of GIRK1. We conclude that CIR/rcKATP-1 is not a subunit of cardiac IKATP and that GIRK1 is the only channel protein coassociating with CIR in heart.

ATP-Binding Cassette Transporters↗
PubMed1995

The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins.

Heart rate is slowed in part by acetylcholine-dependent activation of a cardiac potassium (K+) channel, IKACh. Activated muscarinic receptors stimulate IKACh via the G-protein beta gamma-subunits. It has been assumed that the inwardly rectifying K(+)-channel gene, GIRK1, alone encodes IKACh. It is now shown that IKACh is a heteromultimer of two distinct inwardly rectifying K(+)-channel subunits, GIRK1 and a newly cloned member of the family, CIR.

Amino Acid Sequence↗
PubMed1995

Calcium release from the nucleus by InsP3 receptor channels.

The nucleus is surrounded by a double membrane separating it from the cytoplasm. The perinuclear space is continuous with endoplasmic reticulum, and the nuclear outer membrane shares many features with the reticular membrane. We now show that inositol 1,4,5-trisphosphate (InsP3) receptors associated with the nucleus release Ca2+ from isolated Xenopus laevis oocyte nuclei. Electrophysiological measurements of the intracellular InsP3 receptor in its native membrane have not been possible on the fine filamentous endoplasmic reticulum. In this paper, we directly measure InsP3-dependent receptor channels in isolated nuclei. The nuclear InsP3 receptor is activated by InsP3 and modulated by Ca2+. The channel is weakly regulated by ATP, is mildly voltage dependent, and has a greater conductance with monovalent cations than with divalent cations.

Adenosine Triphosphate↗
PubMed1995

G-protein regulation of ion channels.

Even though the vast majority of ion channels are regulated by voltage, extracellular ligands, phosphorylation, intracellular ions, or a combination of these influences, probably only a handful of ion channels are regulated by direct interaction with activated G proteins. Although results from electrophysiological studies of some channels are consistent with the hypothesis of regulation via direct physical interactions with G proteins, strong biochemical evidence for such interactions is still lacking. In most cases, such evidence has been difficult to obtain because ion channels are present at very low abundances in cell membranes, or because the molecular identity of the channel is unknown. The recent cloning of members of the inwardly rectifying K+ channel and voltage-gated Ca2+ channel families should facilitate the rigorous study of the putative interactions between G proteins and ion channels.

Animals↗
PubMed1995

Ion channel regulation by G proteins.

Ion channels are poised uniquely to initiate, mediate, or regulate such distinct cellular activities as action potential propagation, secretion, and gene transcription. In retrospect, it is not surprising that studies of ion channels have revealed considerable diversities in their primary structures, regulation, and expression. From a functional standpoint, the various mechanisms coopted by cells to regulate channel activity are particularly fascinating. Extracellular ligands, membrane potential, phosphorylation, ions themselves, and diffusible second messengers are all well-established regulators of ion channel activity. Heterotrimeric GTP-binding proteins (G proteins) mediate many of these types of ion channel regulation by stimulating or inhibiting phosphorylation pathways, initiating intracellular cascades leading to elevation of cytosolic Ca2+ or adenosine 3',5'-cyclic monophosphate levels, or by generating various lipid-derived compounds. In some cases, it seems that activated G protein subunits can interact directly with ion channels to elicit regulation. Although there is currently little direct biochemical evidence to support such a mechanism, it is the working hypothesis for the most-studied G protein-regulated ion channels.

Action Potentials↗
PubMed1995

Developmental regulation of TCR-CD3-dependent [Ca2+]i responses of individual normal and pp59fyn-deficient T lymphocytes.

The aim of this study was to ascertain whether different types of T-cell receptor (TCR)-mediated [Ca2+]i signals could begin to explain the different cellular responses of mature and immature T cells to ligation of the TCR-CD3 complex. Using a digital fluorescence imaging system, we measured and compared [Ca2+]i of individual cells from immature and mature murine T-cell populations following application of CD3-epsilon monoclonal antibody (mAb). Our approach revealed distinctions among developmental subsets which were not seen by previous measurements of [Ca2+]i in bulk cell populations. The CD3-mediated [Ca2+]i responses of individual thymocytes were very complex. Latencies to peak [Ca2+]i varied greatly among thymocytes, but the responses of splenic T cells were synchronized, novel evidence that the timing of [Ca2+]i responses may be an important informative parameter for TCR-CD3 signalling. In addition, among cells responding to CD3 mAb, higher peak [Ca2+]i responses correlated with maturity (CD4+ CD8+ thymocytes < single-positive thymocytes < splenic T cells). Examination of cells from pp59fyn-deficient mice showed that pp59fyn deficiency affects the amplitude and probability, but not the latency or synchrony, of CD3-mediated [Ca2+]i responses of CD4+ CD8+ and CD4+ CD8- thymocytes. All subsets showed equivalent receptor-independent mobilization of [Ca2+]i. These developmentally distinct [Ca2+]i features most probably reflect meaningful developmental changes in how the TCR-CD3 complex couples to intracellular signalling machinery including pp59fyn. By clearly showing how [Ca2+]i responses change during development, these results support the hypothesis that distinctive types of [Ca2+]i responses drive thymocyte differentiation.

Animals↗
PubMed1994

Recombinant G-protein beta gamma-subunits activate the muscarinic-gated atrial potassium channel.

Acetylcholine activates inwardly rectifying potassium channels (IK.ACh) in the heart through muscarinic receptor binding and activation of pertussis-toxin-sensitive G proteins. Experiments showing that only the beta gamma-subunit (G beta gamma) activates IK.ACh (ref. 4) were challenged by reports that only the activated alpha-subunit (G alpha) was effective. Here we examine IK.ACh regulation using purified brain and recombinant G-protein subunits. Six recombinant G beta gamma-subunits activated IK.ACh with apparent half-maximal activation concentrations of 3-30 nM. Activation of IK.ACh by recombinant G alpha-GTP gamma S was observed, but this was probably due to release of GTP gamma S from the protein. Importantly, IK.ACh activity elicited by GTP gamma S was inhibited by purified brain and recombinant G alpha-GDP, suggesting that native G beta gamma plays a major role in this pathway. We conclude that G beta gamma is a primary regulator of IK.ACh activity.

Acetylcholine↗
PubMed1994

Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICln.

Cells maintain control of their volume by the passage of KCl and water across their membranes, but the regulatory proteins are unknown. Expression in Xenopus oocytes of a novel protein, pICln, activated a chloride conductance. We have cloned analogs of pICln from rat heart and Xenopus ovary. pICln was identified as an abundant soluble cytosolic protein (approximately 40 kd) that does not immunolocalize with the plasma membrane. pICln was found in epithelial and cardiac cells, brain, and Xenopus oocytes, forming complexes with soluble actin and other cytosolic proteins. Monoclonal antibodies recognizing pICln blocked activation of a native hypotonicity-induced chloride conductance (ICl.swell) in Xenopus oocytes, suggesting that pICln may link actin-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. The high degree of sequence conservation and widespread expression of pICln suggest that it is an important element in cellular volume regulation.

Actins↗
PubMed1994

Calcium channels activated by depletion of internal calcium stores in A431 cells.

Depletion of intracellular calcium stores induces transmembrane Ca2+ influx. We studied Ca(2+)- and Ba(2+)-permeable ion channels in A431 cells after store depletion by dialysis of the cytosol with 10 mM BAPTA solution. Cell-attached patches of cells held at low (0.5 microM) external Ca2+ exhibited transient channel activity, lasting for 1-2 min. The channel had a slope conductance of 2 pS with 200 mM CaCl2 and 16 pS with 160 mM BaCl2 in the pipette. Channel activity quickly ran down in excised inside-out patches and was not restored by InsP3 and/or InsP4. Thapsigargin induced activation in cells kept in 1 mM external Ca2+ after BAPTA dialysis. These channels represent one Ca2+ entry pathway activated by depletion of internal calcium stores and are clearly distinct from previously identified calcium repletion currents.

Barium Compounds↗