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Biomedical subjects

D E Clapham

Publications and source records attributed to D E Clapham.

At least 37 records · Page 2Linked to original sources
PubMed1999

GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels.

GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (IKACh) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein Gbetagamma subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique ( approximately 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta374-501 resulted in an intracellular GIRK1 subunit that produced normal IKACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of IKACh in vivo.

Animals↗
PubMed1999

Conformational changes of the in situ nuclear pore complex.

By bridging the double membrane separating the cell nucleus and cytoplasm, nuclear pore complexes (NPCs) are crucial pathways for the exchange of ions, proteins, and RNA between these two cellular compartments. A structure in the central lumen of the NPC, called the nuclear transport protein, central granule, or nuclear plug, appeared to gate diffusion of intermediate-sized molecules (10-40 kDa) across the nuclear membranes. Visualization of the NPC required drying and fixation of the specimen for electron and atomic force microscopy (AFM), a requirement that has raised doubts about the physiological relevance of the observation. Here we present AFM images of the outer nuclear membranes and NPCs of Xenopus laevis oocytes under more physiological conditions. Measured under a variety of Ca2+ depletion conditions, the central granule appeared to occupy and occlude the lumen of the pore in >80% of NPCs compared to <10% in controls. In a few instances images were obtained of the same NPCs as the solution was changed from control saline to store depletion conditions, and finally to store repletion conditions. We conclude that the central lumen of the nuclear pore complex undergoes a conformational change in response to depletion of nuclear cisternal Ca2+ levels.

Adenosine Triphosphate↗
PubMed1999

Gbetagamma binding increases the open time of IKACh: kinetic evidence for multiple Gbetagamma binding sites.

IKACh is an inwardly rectifying potassium channel that plays an important role in the regulation of mammalian heart rate. IKACh is activated by direct interaction with Gbetagamma subunits of pertussis toxin-sensitive heterotrimeric G-proteins. The stoichiometry of the Gbetagamma/channel complex is currently unknown, and kinetic analysis of the channel behavior has led to conflicting conclusions. Here, we analyze the kinetics of the native IKACh channel in inside-out cardiomyocyte patches activated directly by Gbetagamma. We conclude that the channel has at least two open states and that binding of Gbetagamma prolongs its mean open time duration. These findings imply the existence of at least two binding sites on the channel complex for Gbetagamma. We also show that the duration of the channel opening is negatively correlated with the duration of subsequent channel closing, which further constrains the possible kinetic models. A simple qualitative model describing the kinetic behavior of IKACh is presented.

Animals↗
PubMed1999

Molecular determinants for subcellular localization of PSD-95 with an interacting K+ channel.

Ion channels and PSD-95 are colocalized in specific neuronal subcellular locations by an unknown mechanism. To investigate mechanisms of localization, we used biolistic techniques to express GFP-tagged PSD-95 (PSD-95:GFP) and the K(+)-selective channel Kv1.4 in slices of rat cortex. In pyramidal cells, PSD-95:GFP required a single PDZ domain and a region including the SH3 domain for localization to postsynaptic sites. When transfected alone, PSD-95:GFP was present in dendrites but absent from axons. When cotransfected with Kv1.4, PSD-95:GFP appeared in both axons and dendrites, while Kv1.4 was restricted to axons. When domains that mediate the interaction of Kv1.4 and PSD-95 were disrupted, Kv1.4 localized nonspecifically. Our results provide evidence that Kv1.4 itself may determine its subcellular location, while an associated MAGUK protein is a necessary but not sufficient cofactor.

Animals↗
PubMed1999

Active nuclear import and export is independent of lumenal Ca2+ stores in intact mammalian cells.

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (approximately 10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

Calcium↗
PubMed1999

pICln inhibits snRNP biogenesis by binding core spliceosomal proteins.

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.

Animals↗
PubMed1998

Identification of native atrial G-protein-regulated inwardly rectifying K+ (GIRK4) channel homomultimers.

G-protein-regulated inwardly rectifying K+ (GIRK) channels play critical inhibitory roles throughout the nervous system, heart, and pancreas. They are believed to be heterotetramers consisting of GIRK1 (Kir3.1) and either GIRK2 (Kir3.2), GIRK3 (Kir3.3), or GIRK4 (Kir3.4) subunits. The GIRK1 subunit is hypothesized to be critical to form GIRK channels with normal channel kinetics based on heterologous expression studies. However, GIRK2 and GIRK3 proteins are present in areas of the brain where no GIRK1 has been detected. Here we demonstrate that GIRK tetramers lacking GIRK1 can be purified from bovine heart atria. We have found that only half of GIRK4 is purified as the GIRK1-GIRK4 heterotetramer, whereas the remaining GIRK4 forms a high molecular weight, SDS-resistant complex that does not contain GIRK1. These GIRK4 complexes, most likely GIRK4 homotetramers, were previously not seen because of their aberrant migration on SDS-polyacrylamide gels. We propose that all of GIRK1 and half of GIRK4 proteins in atria combine to form the heterotetramer IKACh, whereas the remaining GIRK4 forms a novel tetrameric complex. GIRK4 homotetramers form channels with unusual single channel behavior, and their contribution to native currents requires further investigation.

Animals↗
PubMed1998

Gbeta binding to GIRK4 subunit is critical for G protein-gated K+ channel activation.

The cardiac G protein-gated K+ channel, IKACh, is directly activated by G protein beta gamma subunits (Gbeta gamma). IKAChis composed of two inward rectifier K+ channel subunits, GIRK1 and GIRK4. Gbeta gamma binds to both GIRK1 and GIRK4 subunits of the heteromultimeric IKACh. Here we delineate the Gbeta gamma binding regions of IKACh by studying direct Gbeta gamma interaction with native purified IKACh, competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in Gbeta gamma binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209-225 or 226-245, effectively competed for Gbeta gamma binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting Gbeta gamma binding and Gbeta gamma activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226-245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of Gbeta gamma binding to IKACh and Gbeta gamma activation of GIRK1/mutant GIRK4 channels. We conclude from this data that Gbeta gamma binding to GIRK4 is critical for IKACh activation.

Amino Acid Sequence↗
PubMed1998

pICln binds to a mammalian homolog of a yeast protein involved in regulation of cell morphology.

Since its cloning and tentative identification as a chloride channel, the function of the pICln protein has been debated. Although there is no consensus regarding the specific function of pICln, it was suggested to play a role, directly or indirectly, in the function of a swelling-induced chloride conductance. Previously, the protein was shown to exist in several discrete protein complexes. To determine the function of the protein, we have begun the systematic identification of all proteins to which it binds. Here we show that four proteins firmly bind to pICln and identify the 72-kDa pICln-binding protein by affinity purification and peptide microsequencing. The interaction between this protein and pICln was verified several ways, including the extraction of several pICln clones from a cDNA library using the 72-kDa protein as a bait in a yeast two-hybrid screen. The protein is homologous to the yeast Skb1 protein. Skb1 interacts with Shk1, a homolog of the p21(Cdc42/Rac)-activated protein kinases (PAKs). The known involvement of PAKs in cytoskeletal rearrangement suggests that pICln may be linked to a system regulating cell morphology.

Amino Acid Sequence↗
PubMed1998

Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh.

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.

Animals↗
PubMed1998

Abnormal heart rate regulation in GIRK4 knockout mice.

Acetylcholine (ACh) released from the stimulated vagus nerve decreases heart rate via modulation of several types of ion channels expressed in cardiac pacemaker cells. Although the muscarinic-gated potassium channel I(KACh) has been implicated in vagally mediated heart rate regulation, questions concerning the extent of its contribution have remained unanswered. To assess the role of I(KACh) in heart rate regulation in vivo, we generated a mouse line deficient in I(KACh) by targeted disruption of the gene coding for GIRK4, one of the channel subunits. We analyzed heart rate and heart rate variability at rest and after pharmacological manipulation in unrestrained conscious mice using electrocardiogram (ECG) telemetry. We found that I(KACh) mediated approximately half of the negative chronotropic effects of vagal stimulation and adenosine on heart rate. In addition, this study indicates that I(KACh) is necessary for the fast fluctuations in heart rate responsible for beat-to-beat control of heart activity, both at rest and after vagal stimulation. Interestingly, noncholinergic systems also appear to modulate heart activity through I(KACh). Thus, I(KACh) is critical for effective heart rate regulation in mice.

Adenosine↗
PubMed1998

A novel inward rectifier K+ channel with unique pore properties.

We have cloned a novel K+-selective, inward rectifier channel that is widely expressed in brain but is especially abundant in the Purkinje cell layer of the cerebellum and pyramidal cells of the hippocampus. It is also present in a wide array of tissues, including kidney and intestine. The channel is only 38% identical to its closest relative, Kir1.3 (Kir1-ATP-regulated inward rectifier K+ [ROMK] family) and displays none of the functional properties unique to the ROMK class. Kir7.1 has several unique features, including a very low estimated single channel conductance (approximately 50 fS), low sensitivity to block by external Ba2+ and Cs+, and no dependence of its inward rectification properties on the internal blocking particle Mg2+. The unusual pore properties of Kir7.1 seem to be explained by amino acids in the pore sequence that differ from corresponding conserved residues in all other Kir channel proteins. Replacement of one of these amino acids (Met-125) with the Arg absolutely conserved in all other Kir channels dramatically increases its single channel conductance and Ba2+ sensitivity. This channel would provide a steady background K+ current to help set the membrane potential in cells in which it is expressed. We propose that the novel channel be assigned to a new Kir subfamily, Kir7.1.

Animals↗
PubMed1998

NMDA receptors amplify calcium influx into dendritic spines during associative pre- and postsynaptic activation.

Long-term potentiation (LTP) of synaptic strength can be induced by synchronous pre- and postsynaptic activation, and a rise in postsynaptic calcium is essential for induction of LTP. Calcium can enter through both voltage-dependent Ca2+ channels and NMDA-type glutamate receptors, but the relative contributions of these pathways is not known. We have examined this issue in layer V cortical pyramidal neurons, using focal flash photolysis of caged glutamate to mimic synaptic input and two-photon, laser-scanning microscopy to measure calcium levels in dendritic spines. Most of the calcium entry in response to glutamate alone was via voltage-dependent Ca2+ channels, and NMDA receptors accounted for less than 20% of total Ca2+ entry. When glutamate was paired with postsynaptic action potentials, however, the NMDA-receptor-dependent component was selectively amplified. The same is likely to occur during paired physiological pre- and postsynaptic activation, providing a mechanism for the input specificity and Hebbian behavior of LTP.

Action Potentials↗
PubMed1998

At last, the structure of an ion-selective channel.

Ion channels allow specific ions to flow across the cell membrane at rates of ten million ions per second with error rates of only 1 in 1,000. The structure of a protein that accomplishes this feat has now been discovered.

Bacterial Proteins↗