Biomedical subjects

K Wickman

Publications and source records attributed to K Wickman.

At least 19 recordsLinked to original sources

PubMed2001

Evaluation of the role of I(KACh) in atrial fibrillation using a mouse knockout model.

OBJECTIVES: We sought to study the role of I(KACh) in atrial fibrillation (AF) and the potential electrophysiologic effects of a specific I(KACh) antagonist. BACKGROUND: I(KACh) mediates much of the cardiac responses to vagal stimulation. Vagal stimulation predisposes to AF, but the specific role of I(KACh) in the generation of AF and the electrophysiologic effects of specific I(KACh) blockade have not been studied. METHODS: Adult wild-type (WT) and I(KACh)-deficient knockout (KO) mice were studied in the absence and presence of the muscarinic receptor agonist carbachol. The electrophysiologic features of KO mice were compared with those of WT mice to assess the potential effects of a specific I(KACh) antagonist. RESULTS: Atrial fibrillation lasting for a mean of 5.7+/-11 min was initiated in 10 of 14 WT mice in the presence of carbachol, but not in the absence of carbachol. Atrial arrhythmia could not be induced in KO mice. Ventricular tachyarrhythmia could not be induced in either type of mouse. Sinus node recovery times after carbachol and sinus cycle lengths were shorter and ventricular effective refractory periods were greater in KO mice than in WT mice. There was no significant difference between KO and WT mice in AV node function. CONCLUSIONS: Activation of I(KACh) predisposed to AF and lack of I(KACh) prevented AF. It is likely that I(KACh) plays a crucial role in the generation of AF in mice. Specific I(KACh) blockers might be useful for the treatment of AF without significant adverse effects on the atrioventricular node or the ventricles.

Animals↗

PubMed2000

Functional and biochemical evidence for G-protein-gated inwardly rectifying K+ (GIRK) channels composed of GIRK2 and GIRK3.

G-protein-gated inwardly rectifying K(+) (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K(+) channels, they drive the transmembrane potential toward E(K) when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo- or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an approximately 5-fold lower sensitivity to activation by Gbetagamma. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.

Animals↗

PubMed2000

Brain localization and behavioral impact of the G-protein-gated K+ channel subunit GIRK4.

Neuronal G-protein-gated potassium (K(G)) channels are activated by several neurotransmitters and constitute an important mode of synaptic inhibition in the mammalian nervous system. K(G) channels are composed of combinations of four subunits termed G protein-gated inwardly rectifying K(+) channels (GIRK). All four GIRK subunits are expressed in the brain, and there is a general consensus concerning the expression patterns of GIRK1, GIRK2, and GIRK3. The localization pattern of GIRK4, however, remains controversial. In this study, we exploit the negative background of mice lacking a functional GIRK4 gene to identify neuronal populations that contain GIRK4 mRNA. GIRK4 mRNA was detected in only a few regions of the mouse brain, including the deep cortical pyramidal neurons, the endopiriform nucleus and claustrum of the insular cortex, the globus pallidus, the ventromedial hypothalamic nucleus, parafascicular and paraventricular thalamic nuclei, and a few brainstem nuclei (e.g., the inferior olive and vestibular nuclei). Mice lacking GIRK4 were viable and appeared normal and did not display gross deficiencies in locomotor activity, visual tasks, and pain perception. Furthermore, GIRK4-deficient mice performed similarly to wild-type controls in the passive avoidance paradigm, a test of aversive learning. GIRK4 knock-out mice did, however, exhibit impaired performance in the Morris water maze, a test of spatial learning and memory.

Animals↗

PubMed2000

ICln is essential for cellular and early embryonic viability.

pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens. The physiological functions of pICln remain to be established. To address this question, we disrupted the ICln gene in embryonic stem cells. We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5). Furthermore, we found that ICln is essential for embryonic stem cell viability. Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling. pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis. Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.

3T3 Cells↗

PubMed1999

Structure, G protein activation, and functional relevance of the cardiac G protein-gated K+ channel, IKACh.

The muscarinic-gated atrial potassium channel IKACh has been well characterized functionally, and has been an excellent model system for studying G protein/effector interactions. Complementary DNAs encoding the composite subunits of IKACh have been identified, allowing direct probing of structural and functional features of the channel. Here, we highlight recent approaches taken in our laboratory to determine the oligomeric structure of native cardiac IKACh, the mechanism of activation of IKACh by G proteins, and the relevance of IKACh to cardiac physiology.

Amino Acid Sequence↗

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

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↗

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

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

Using knockout and transgenic mice to study neurophysiology and behavior.

Reverse genetics, in which detailed knowledge of a gene of interest permits in vivo modification of its expression or function, provides a powerful method for examining the physiological relevance of any protein. Transgenic and knockout mouse models are particularly useful for studies of complex neurobiological problems. The primary aims of this review are to familiarize the nonspecialist with the techniques and limitations of mouse mutagenesis, to describe new technologies that may overcome these limitations, and to illustrate, using representative examples from the literature, some of the ways in which genetically altered mice have been used to analyze central nervous system function. The goal is to provide the information necessary to evaluate critically studies in which mutant mice have been used to study neurobiological problems.

Animals↗

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↗

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

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↗

PubMed1992

New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.

Amino Acid Sequence↗

PubMed1986

Clinical significance of nontuberculous mycobacteria. A bacteriological survey of Swedish strains isolated between 1973 and 1981.

An account is rendered of mycobacteria other than Mycobacterium tuberculosis isolated from specimens sent for culture during 9 years. The possible etiological significance of these bacteria was estimated from clinical data and the degree of bacterial growth. Pulmonary infection was the most common nontuberculous manifestation in the adults. In about 50% of the patients with slowly growing mycobacteria in their respiratory tract, these bacteria were considered to be an etiological factor. This applied to the species of the M. avium-intracellulare (MAI) complex and M. xenopi. M. Kansasii, a common cause of lung disease in many other countries, was rarely isolated. The corresponding figure for the rapidly growing mycobacteria belonging to the M. fortuitum-chelonei complex was 15%. These patients were all severely debilitated by other diseases. Lymphadenitis was the most common nontuberculous infection in children, but occurred only in children not vaccinated with BCG. Bacteria of the MAI complex was the predominating cause. Lung infection caused by nontuberculous mycobacteria did not occur in children.

Adolescent↗