Biomedical subjects

P A Shelton

Publications and source records attributed to P A Shelton.

10 recordsLinked to original sources

PubMed1998

The dependence of Ag+ block of a potassium channel, murine kir2.1, on a cysteine residue in the selectivity filter.

Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.

Amino Acid Sequence↗

PubMed1996

The use of the rat LAP LCR and promoter for the high-level constitutive expression of K+ channel cDNAs in a rat liver cell line.

Locus control regions (LCRs) are cis-acting elements that confer position-independent and copy-number-dependent expression upon associated genes in transgenic mice. Here we show the second example of the use of an LCR (the rat LAP LCR) in a stable expression vector system, used here in conjunction with the rat liver (NRLM) cell line. Non-transfected NRLM cells are electrically silent and highly suitable for patch clamp electrophysiology. We report reliable constitutive expression from two different K+ channel cDNAs; the voltage-gated rat clone Kv3.4 and the inward rectifier mouse clone Kir2.1. We further show that constitutive expression levels are stable for at least 8 weeks from initial recording.

Animals↗

PubMed1996

The role of a single aspartate residue in ionic selectivity and block of a murine inward rectifier K+ channel Kir2.1.

1. The effects of Rb+ and Cs+ as blocking ions were investigated on wild-type and mutant forms of the inward rectifier K+ channel, IRK1 (Kir2.1). 2. In wild-type channels, Rb+ blockage was voltage dependent, increasing and then falling with increasing hyperpolarization. 3. Rb+ blockage was abolished by replacing Asp172 in the M2 domain of the pore-forming subunit by Asn, but was re-established by a change to Gln, narrowing the pore. Blocking affinity was reduced in D172Q, and was also reduced by replacing Gly168 in M2 by Ala. 4. Cs+ blockage was also abolished in D172N but was re-established in D172Q. 5. There appears to be a balance between charge and pore size in determining whether ions block or permeate. A major part of the selectivity of Kir2.1 is associated with Asp172 in the M2 domain.

Animals↗

PubMed1994

A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane-spanning alpha-helix of the murine inward rectifier, IRK1, by site-directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.

Amino Acid Sequence↗

PubMed1994

The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+.

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole-cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e-fold as the driving force on K+(V-EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current-voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.

Animals↗

PubMed1994

Apperceptive visual agnosia: a case study.

A man with an infarction of his inferior temporal and occipital association cortex bilaterally, which spared primary visual cortex, had impaired visual recognition of objects, faces, colors, words, and gestures. Analysis of visual function indicated that the recognition failures resulted from an agnosia, rather than elemental visual impairment. Whereas his impairment of gesture recognition appeared to be related to an associative agnosia, his inability to recognize objects was related to an apperceptive agnosia. There may be four subtypes of apperceptive agnosia: one where the internal object representations or structural descriptions are impaired, another where an adequate percept cannot be derived, a third where the internal referent and percept are dissociated, and a fourth where both levels are impaired. Our patient demonstrated a failure to relate individual elements to the whole, a failure to integrate multiple elements, and a reliance on global perception. He had normal object imagery. These results suggest that, whereas internal representations were intact, he was unable to form adequate perceptual representations.

Aged↗

PubMed1993

The Ustilago maydis nar1 gene encoding nitrate reductase activity: sequence and transcriptional regulation.

The nar1 gene was cloned from Ustilago maydis and the 908-amino-acid (aa) sequence of the encoded protein found to have strong identities with other nitrate reductases from fungi and plants. This was especially so in three domains which define enzyme cofactor-binding sites. The gene was isolated alone and in association with the nir1 gene, suggesting that the two genes are closely linked on the chromosome. The phenotype of a strain in which nar1 had been disrupted was consistent with the only role of nar1 being in nitrate reduction. Nitrate ions induced a 90-fold increase in nar1 transcript levels, while ammonium ions repressed transcript levels.

Amino Acid Sequence↗

PubMed1993

Regulated expression of K+ channel genes in electrically silent mammalian cells by linkage to beta-globin gene-activation elements.

Fundamental studies of the mechanism of human beta-like globin gene-expression have identified DNA sequences (locus control regions or LCRs) which directly influence the specific activation of beta-globin genes in erythroid cells. Here we report the first applications of LCR-mediated gene-activation to stable electrophysiological expression of several homomultimeric ion channel proteins. We describe expression driven from a native K+ channel gene promoter region, contiguous to an intronless coding sequence, within a single excised human genomic DNA fragment. In addition, cDNAs encoding both inactivating and non-inactivating mammalian K+ currents have been expressed by insertion between the promoter and second intron of the human beta-globin gene. Expression levels are characteristically independent of integration position and are proportional to LCR-gene copy number, a parameter specific for each clonal cell line. K+ channel expression is inducible by erythroid differentiation and has been demonstrated by electrophysiological recordings of cells taken directly from culture.

Amino Acid Sequence↗

PubMed1991

Ideomotor apraxia in Huntington's disease.

The pattern of movement errors in ideomotor apraxia suggests an abnormality in selection and sequencing of component movements. Individuals with Huntington's disease were evaluated prospectively for the presence of apraxia, and aspects of motor and cognitive function were correlated with apraxic errors. Based on a conservative apraxia rating, ideomotor apraxia occurred in three (33%) of nine patients with a mean duration of disease of 10.4 years. Only two (22%) individuals made no apraxic errors, however, and the group as a whole made apraxic errors in 26% of movements. Apraxia was associated with errors in imitation of nonsymbolic movements but not with errors in recognition of gestures. It correlated significantly with duration of disease and with progressive abnormalities of posture but not with other individual aspects of elementary motor or cognitive function. These associations indicate that apraxia in Huntington's disease may be due primarily to involvement of subcortical motor structures rather than cerebral cortex.

Adult↗

PubMed1990

Peripersonal and vertical neglect.

When bisecting radial lines visually, normal subjects err towards distant peripersonal space, and when bisecting vertical lines visually, they err towards upper vertical space. In contrast, when bisecting lines under tactile-proprioceptive guidance, subjects err towards near peripersonal space, suggesting that normally attention is preferentially distributed away from the body during visual exploration but distributed towards the body during tactile exploration. A patient with ischaemic lesions, however, involving both inferior temporal lobes neglected far peripersonal and upper vertical space. He also demonstrated a motor bias away from the neglected space. These findings suggest that in man attention is spatially directed in three orthogonal axes (horizontal, vertical and radial), that attention may normally be unequally distributed in each of these axes, and that neglect may occur in not only the horizontal axis but also in the radial and vertical axes.

Adult↗