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Biomedical subjects

A Tinker

Publications and source records attributed to A Tinker.

At least 19 recordsLinked to original sources
PubMed2004

Participation of RGS8 in the ternary complex of agonist, receptor and G-protein.

The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein alpha subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K(+) channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and G(o) alpha(A)-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gbetagamma overexpression. FRET was also seen between RGS8 and Ggamma, demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein alpha subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the 'kinetic efficacy' of G-protein activation.

Cell Line↗
PubMed2002

K(ATP) channel gene expression is induced by urocortin and mediates its cardioprotective effect.

BACKGROUND: Urocortin is a novel cardioprotective agent that can protect cardiac myocytes from the damaging effects of ischemia/reperfusion both in culture and in the intact heart and is effective when given at reperfusion. METHODS AND RESULTS: We have analyzed global changes in gene expression in cardiac myocytes after urocortin treatment using gene chip technology. We report that urocortin specifically induces enhanced expression of the Kir 6.1 cardiac potassium channel subunit. On the basis of this finding, we showed that the cardioprotective effect of urocortin both in isolated cardiac cells and in the intact heart is specifically blocked by both generalized and mitochondrial-specific K(ATP) channel blockers, whereas the cardioprotective effect of cardiotrophin-1 is unaffected. Conversely, inhibiting the Kir 6.1 channel subunit greatly enhances cardiac cell death after ischemia. CONCLUSIONS: This is, to our knowledge, the first report of the altered expression of a K(ATP) channel subunit induced by a cardioprotective agent and demonstrates that K(ATP) channel opening is essential for the effect of this novel cardioprotective agent.

Adenosine Triphosphate↗
PubMed2001

Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca(2+)-independent protein kinase C.

1. Members of the Kir3.0 family of inwardly rectifying K(+) channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by G(betagamma) subunits released in response to G(i/o)-coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the G(q/11)-coupled M(1) and M(3) muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal-type channels stably expressed in the human embryonic kidney cell line HEK293. 2. We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M(1) and M(3) receptors. This contrasts with the simple stimulation seen through activation of M(2) and M(4) receptors. 3. Channel stimulation via M(1) but not M(3) receptors was sensitive to pertussis toxin whereas channel inhibition through both M(1) and M(3) receptors was insensitive. In contrast over-expression of the C-terminus of phospholipase Cbeta1 or a G(q/11)-specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. 4. The inhibitory effects of M(1) and M(3) receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4alphaphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M(3) receptor. 5. The receptor-mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0-669 nM). 6. The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCdelta and PKCepsilon. 7. We also demonstrate that activation of such a pathway via both receptor-mediated and receptor-independent means profoundly attenuated subsequent channel stimulation by G(i/o)-coupled receptors. 8. Our data support a role for a Ca(2+)-independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M(1) and M(3) muscarinic receptor stimulation.

Carbachol↗
PubMed2001

A mechanism for ATP-sensitive potassium channel diversity: Functional coassembly of two pore-forming subunits.

ATP-sensitive potassium channels are an octomeric complex of four pore-forming subunits of the Kir 6.0 family and four sulfonylurea receptors. The Kir 6.0 family consists of two known members, Kir 6.1 and Kir 6.2, with distinct functional properties. The tetrameric structure of the pore-forming domain leads to the possibility that mixed heteromultimers may form. In this study, we examine this by using biochemical and electrophysiological techniques after heterologous expression of these subunits in HEK293 cells. After the coexpression of Kir 6.1 and Kir 6.2, Kir 6.1 can be coimmunoprecipitated with isoform-specific Kir 6.2 antisera and vice versa. Coexpression of SUR2B and Kir 6.2 with Kir 6.1 dominant negatives at a 1:1 expression ratio and vice versa led to a potent suppression of current. Kir 6.1, and Kir 6.2 dominant negative mutants were without effect on an inwardly rectifying potassium channel from a different family, Kir 2.1. Single-channel analysis, after coexpression of SUR2B, Kir 6.1, and Kir 6.2, revealed the existence of five distinct populations with differing single-channel current amplitudes. All channel populations were inhibited by glibenclamide. A dimeric Kir 6.1-Kir 6.2 construct expressed with SUR2B had a single-channel conductance intermediate between that of either Kir 6.2 or Kir 6.1 expressed with SUR2B. In conclusion, Kir 6.1 and Kir 6.2 readily coassemble to produce functional channels, and such phenomena may contribute to the diversity of nucleotide-regulated potassium currents seen in native tissues.

ATP-Binding Cassette Transporters↗
PubMed2001

A spectrum of functional effects for disease causing mutations in the Jervell and Lange-Nielsen syndrome.

OBJECTIVE: Jervell and Lange-Nielsen syndrome (JLNS) is a recessively inherited long QT syndrome (LQTS) characterised by profound sensorineural deafness and predisposition to syncope and sudden cardiac death. Mutation analysis has established the presence of mutations in affected individuals in the genes KCNQ1 and KCNE1: the potassium channel complex responsible for the cardiac I(Ks) current involved in repolarisation of the ventricular action potential. Our objective was to determine the functional effects of disease causing mutations in JLNS. METHODS: In this study we have investigated the electrophysiological effects of eight distinct JLNS mutations after expression of cRNA in Xenopus laevis oocytes. RESULTS: KCNE1 mutant T59P/L60P showed no dominant negative effect and was a pure loss of function mutation. KCNQ1 mutant E261D showed a strong dominant-negative effect. KCNQ1 mutant R243H produced a moderate dominant-negative effect, right shifted the steady-state activation curve and led to an increased deactivation rate. The behaviour of KCNQ1 mutants 572-576del, 1008delC, R518X, Q530X, R594Q depended on the relative quantities of mutant and wild-type proteins (with a weak dominant-negative effect present at 1:3 but not 1:1 injection ratios). These data indicate the presence of an additional assembly domain before S2-S3 and the importance of the S4-S5 region in channel function and gating. CONCLUSIONS: Our data suggest a spectrum of behaviour for disease causing mutations from simple loss of function through to prominent dominant negative behaviour.

Animals↗
PubMed2001

Factors associated with nursing home entry for older people in Taiwan, Republic of China.

Taiwan is facing a rapid change in the composition of its population. As the population ages, a greater demand for long-term care services and, in particular, nursing homes is expected. Before deciding who really needs nursing home care, it is important for policy makers to understand the current pattern of utilisation and what factors are associated with entry. This research assesses the relative importance of predisposing, enabling and need factors that lie behind this. It is based on a survey of elderly people in registered nursing homes, a comparison with a national sample of elderly people in their own homes and interviews with the lucid elderly patients (i.e. could communicate with no problems) and their carers. It was found that nursing home entry was associated with advanced age, gender, educational level and dependency levels of elderly people. After controlling for age, need factors have the greatest impact on admission. Specific medical problems such as cardiovascular, neurological and skeletal muscular diseases were also major contributors. Although most elderly people in Taiwan are cared for in their own homes by their families, under certain circumstances entry to a nursing home seemed inevitable. Decisions about nursing home entry were mainly taken within a family context with adult children being the main players while professionals played a relatively minimal role.

Adaptation, Psychological↗
PubMed2000

Functional expression of the pore forming subunit of the ATP-sensitive potassium channel in Saccharomyces cerevisiae.

We have expressed the pore-forming subunits (Kir 6.1 and Kir 6.2) of the mammalian ATP-sensitive potassium channel in a potassium-transport deficient yeast strain (trk1 trk2). Functional expression of Kir 6.2 and Kir 6.1 can complement growth deficiency weakly and strongly respectively of the yeast strain on low-potassium medium. Mutations of Kir 6.2 that abolish ATP sensitivity (K185Q, I182Q) and enhance trafficking to the plasma membrane surface (Kir 6.2DeltaC36) lead to significantly better growth rescue. Growth rescue of Kir 6.1, Kir 6.2 and the above mutants can be inhibited by pharmacological agents (cesium ions, phentolamine and quinine) known to decrease channel activity by direct interaction with the pore forming subunit. Thus we have developed a system in yeast that can report both loss and gain of function mutations in these subunits and pharmacological interventions.

Adenosine Triphosphate↗
PubMed2000

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel.

Inwardly rectifying potassium (K(+)) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by G(betagamma) subunits released from G protein heterotrimers of the G(i/o) family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein alpha subunits (G(ialpha1-3) and G(oalphaA)) in mediating coupling between various receptor systems (A(1), alpha(2A), D(2S), M(4), GABA(B)1a+2, and GABA(B)1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant G(i/oalpha) subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A(1), alpha(2A)) known to interact with all isoforms, G(ialpha1-3) and G(oalphaA) can all support a significant degree of coupling to Kir3.1+3.2A. The M(4) receptor appears to preferentially couple to G(ialpha2) while another group of receptors (D(2S), GABA(B)1a+2, GABA(B)1b+2) activates the channel predominantly through G(betagamma) liberated from G(oA) heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABA(B)1. Our data reveal selective pathways of receptor activation through different G(i/oalpha) isoforms for stimulation of the G protein-gated inwardly rectifying K(+) channel.

Cell Line↗
PubMed2000

The G protein alpha subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly rectifying K(+) channels.

In neuronal and atrial tissue, G protein-gated inwardly rectifying K(+) channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by Gbetagamma dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G(i/o) family but not receptors coupled to the stimulatory G protein G(s). We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein alpha subunits and chimeras between G(i1) and G(s) indicate a central role for the G protein alpha subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K(+) channels.

Adenosine↗
PubMed2000

Population aspects of the dementias.

In the United Kingdom there are some key facts which have implications for policies and services for people with dementia. The first group are to do with older people and their position in the total population. Overall the population of this country is projected to increase, but only by 0.2%. However, within that the population is ageing, i.e. the proportion of older people in the population is projected to increase and the proportion of very old people in the older population is also projected to increase. Also of significance is that the population of working age is projected to decline. Second gender and marital status are important variables. Third is the need to consider where people live. The two important facts here are that the proportion of people living alone is increasing, and expected to grow further, and family patterns are changing. Both of these will have an effect on who is available to care. But we also need to look at the implications of population change in the EU which have implications for the UK. Some of the implications and issues of these population aspects are then discussed.

Age Factors↗
PubMed2000

Women's health: the unfinished agenda.

Improving women's health and nutrition could save millions of women in developing countries from needless suffering or premature death. Cost-effective health interventions exist to prevent this loss of lives. Complications of pregnancy and childbirth are the leading cause of death and disability among women of reproductive age in developing countries. Malnutrition is a major contributory factor to women's poor health and preventable mortality. Domestic violence, sexual abuse, and female genital mutilation carry a heavy physical and mental toll, as well as constitute an intolerable violation of human rights. Women's health is influenced by complex biological, social, and cultural factors that are highly interrelated. Significant progress can be achieved by strengthening and expanding an essential package of health services for women, improving the policy environment, and promoting more positive attitudes and behavior towards women's health.

Circumcision, Female↗
PubMed2000

Elder abuse: do general practitioners know or care?

A pilot survey in Tower Hamlets, London, indicated that many general practitioners (GPs) might not be recognizing abuse of elderly patients through lack of training. The survey was replicated on a large scale in Birmingham, to allow further analysis. 561 Birmingham GPs were mailed questionnaires and responses from 291 were analysed, providing data from 95% of the practices. The findings were similar to those in Tower Hamlets: just under half had diagnosed elder abuse in the previous year. Regression analysis of the combined data-sets (n = 363) indicated that the strongest factor predicting GP diagnosis of abuse was knowledge of 5 or more risk situations (odds ratio 6.77, 95% confidence interval 4.19, 10.93). The findings of these surveys suggest that research-based education and training would help GPs to become better at identifying and managing elder abuse.

Aged↗
PubMed1999

The molecular assembly of ATP-sensitive potassium channels. Determinants on the pore forming subunit.

ATP-sensitive potassium channels form a link between membrane excitability and cellular metabolism. These channels are important in physiological processes such as insulin release and they are an important site of drug action. They are an octomeric complex comprised of four sulfonylurea receptors, a member of the ATP-binding cassette family of proteins, and four Kir 6.0 subunits from the inward rectifier family of potassium channels. We have investigated the nature of the interaction between SUR1 and Kir 6.2 and the domains on the channel responsible for the biochemical and functional manifestations of coupling. The results point to the proximal C terminus determining biochemical interaction in a region that also largely governs homotypic and heterotypic interaction between different Kir family members. While this domain may be necessary for functional communication between the two proteins, it is not sufficient since relative modifications of either the N or C terminus are able to disrupt many aspects of functional coupling mediated by the sulfonylurea receptor.

ATP-Binding Cassette Transporters↗
PubMed1998

Potassium channels in the vasculature.

Many important cellular functions are regulated by vascular potassium channels, including the resting membrane potential. Recent evidence suggests that the function of these channels is altered in pathophysiological disorders of the cardiovascular system. Using molecular cloning techniques, considerable effort has been made over the past 5 years to elucidate the structure of various types of potassium channels. Several different potassium channel clones have been identified from neuronal and cardiac tissues, although only a few have so far been identified in smooth muscle.

Animals↗