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C M Armstrong

Publications and source records attributed to C M Armstrong.

At least 19 recordsLinked to original sources
PubMed2001

A model for 4-aminopyridine action on K channels: similarities to tetraethylammonium ion action.

We present a model for the action of 4-aminopyridine (4AP) on K channels. The model is closely based on the gating current studies of the preceding paper and has been extended to account for ionic current data in the literature. We propose that 4AP, like tetraethylammonium ion and other quaternary ammonium ions, enters and leaves the channel only when the activation gate is open, a proposal that is strongly supported by the literature. Once in the open channel, 4AP's major action is to bias the activation gate toward the closed conformation by approximately the energy of a hydrogen bond. S4 segment movement, as reflected in gating currents, is almost normal for a 4AP-occupied channel; only the final opening transition is affected. The model is qualitatively the same as the one used for many years to explain the action of quaternary ammonium ions.

4-Aminopyridine↗
PubMed2001

Resolving the gating charge movement associated with late transitions in K channel activation.

We examined the late transitions in the activation sequence of potassium channels by analyzing gating currents of mutant Shaker IR channels and using the potassium channel blocker 4-aminopyridine (4AP). Gating currents were recorded from a double mutant of Shaker that was nonconducting (W434F mutation) and had the late gating transitions shifted to the right on the voltage axis (L382C mutation), thus separating the late transitions from the early ones. 4AP applied to the double mutant blocked the final transition and made possible novel observations of the isolated intermediate transitions, the ones that immediately precede the final opening of the channel. These transitions, which have not been well characterized previously, produce a distinct fast component in the gating current tails. Two intermediate transitions contribute to the fast component and carry 23% of the total gating charge. The effect of 4AP is well modeled as a selective block of the final gating transition, which opens the channel. The final transition contributes approximately 5% of the total gating charge.

4-Aminopyridine↗
PubMed2001

Mechanism underlying slow kinetics of the OFF gating current in Shaker potassium channel.

Based on the structure of the KcsA potassium channel, the Shaker K+ channel is thought to have, near the middle of the membrane, a cavity that can be occupied by a permeant or a blocking cation. We have studied the interaction between cations in the cavity and the activation gate of the channel, using a set of monovalent cations together with Shaker mutants that modify the structure of the cavity. Our results show that reducing the size of the side chain at position 470 makes it possible for the mutant channel, unlike native Shaker, to close with tetraethylammonium (TEA+) or the long-chain TEA-derivative C10+ trapped inside the channel. Neither I470 mutants nor Shaker can close when N-methyl-glucamine (NMG+) is in the channel, even though this ion is smaller than C10+. Apparently, the carbohydrate side chain of NMG+ prevents gate closing. Gating currents recorded from Shaker and I470C were measured in the presence of different intracellular cations to further analyze the interaction of cations with the gate. Our results suggest that the cavity in Shaker is so small that even permeant cations like Rb+ or Cs+ must leave the cavity before the channel gate can close.

Alanine↗
PubMed2001

Revisiting the role of Ca2+ in Shaker K+ channel gating.

Shaker K+ channels were expressed in outside-out macropatches excised from Xenopus oocytes, and the effects on gating of removal of extracellular Ca2+ were examined in the complete absence of intracellular divalent cations. Removal of extracellular Ca2+ by perfusion with EDTA-containing solution caused a small negative shift in the channel's voltage-activation curve and led to an increased nonselective leak, but did not otherwise alter or disrupt the channels. The results contradict the proposal that Ca2+ is an essential component required for maintenance of ion selectivity and proper gating of Kv-type K+ channels. The large nonselective leak in Ca2+-free conditions was found to be a patch-seal phenomenon related to F- ion in the recording pipette.

Animals↗
PubMed2000

Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase.

Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

Acetylation↗
PubMed2000

Evidence for genetic heterogeneity in families with congenital motor nystagmus (CN).

Congenital motor nystagmus (CN) is a relatively common genetic disorder (approximately 1 in 1500) characterized by bilateral involuntary ocular oscillations, with onset occurring within the first six months of life. To date, three loci associated with CN have been mapped to chromosomes 6p12, Xp11.4-p11.3, and Xq26-q27. We analyzed five pedigrees segregating for CN. Mapping studies using markers in these three regions showed that only one pedigree exhibited suggestive linkage with a lod score of 2.08, straight theta=0.0, at chromosome Xp11. This pedigree had both affected male and female members, with two unaffected obligate female carriers. The remaining four pedigrees did not exhibit evidence of linkage for any of the three chromosome locations. Three of the pedigrees, Pedigrees 2, 4, and 5, exhibited several instances of male-to-male transmission, excluding X-linkage, and exhibited a lod score of -3.82, straight theta=0.0, for marker D6S459 located at 6p12, thus excluding the chromosome 6 locus. This provides evidence for at least a fourth locus associated with CN.

Chromosome Mapping↗
PubMed1999

Calcium block of Na+ channels and its effect on closing rate.

Calcium ion transiently blocks Na+ channels, and it shortens the time course for closing of their activation gates. We examined the relation between block and closing kinetics by using the Na+ channels natively expressed in GH3 cells, a clonal line of rat pituitary cells. To simplify analysis, inactivation of the Na+ channels was destroyed by including papain in the internal medium. All divalent cations tested, and trivalent La3+, blocked a progressively larger fraction of the channels as their concentration increased, and they accelerated the closing of the Na+ channel activation gate. For calcium, the most extensively studied cation, there is an approximately linear relation between the fraction of the channels that are calcium-blocked and the closing rate. Extrapolation of the data to very low calcium suggests that closing rate is near zero when there is no block. Analysis shows that, almost with certainty, the channels can close when occupied by calcium. The analysis further suggests that the channels close preferentially or exclusively from the calcium-blocked state.

Animals↗
PubMed1999

Distinguishing surface effects of calcium ion from pore-occupancy effects in Na+ channels.

The effects of calcium ion on the Na+ activation gate were studied in squid giant axons. Saxitoxin (STX) was used to block ion entry into Na+ channels without hindering access to the membrane surface, making it possible to distinguish surface effects of calcium from pore-occupancy effects. In the presence of STX, gating kinetics were measured from gating current (Ig). The kinetic effects of external calcium concentration changes were small when STX was present. In the absence of STX, lowering the calcium concentration (from 100 to 10 mM) slowed the closing of Na+ channels (measured from INa tails) by more than a factor of 2. Surprisingly, the voltage sensitivity of closing kinetics changed with calcium concentration, and it was modified by STX. Voltage sensitivity apparently depends in part on the ability of calcium to enter and block the channels as voltage is driven negative. In external medium with no added calcium, INa tail current initially increases in amplitude severalfold with the relief of calcium block, then progressively slows and gets smaller, as calcium diffuses out of the layers investing the axon. INa tails seen just before the current disappears suggest that closing in the absence of channel block is very slow or does not occur. INa amplitude and kinetics are completely restored when calcium is returned. The results strongly suggest that calcium occupancy is a requirement for channel closing and that nonoccupied channels fold reversibly into a nonfunctional conformation.

Animals↗
PubMed1998

Multiplexed short tandem repeat polymorphisms of the Weber 8A set of markers using tailed primers and infrared fluorescence detection.

Short tandem repeat polymorphism (STRP) markers have become important reagents for mapping genetic diseases. These markers are available as screening sets, which are located in all chromosomes at discrete intervals, allowing the entire genome to be analyzed. Mapping studies that include many individuals in the analysis necessitate the production of large numbers of genotypes. In an effort to increase the efficiency and lower the cost of using these STRP screening sets, we have divided the amplification primers of the Weber 8A screening set into groups that can be amplified in single polymerase chain reaction (PCR) amplification reactions, resulting in a reduction of both time and cost. Fluorescently-labeled amplification products were produced using a three primer reaction. The forward STRP amplification primer for each marker contained a 19 bp sequence at the 5' end. A fluorescently-labeled primer, with a sequence identical to the 19 bp tail, was added to the amplification reaction as the sole source of fluorescent label. The STRP banding pattern is detected using an automated fluorescent DNA sequencer. Use of this multiplexed genomic screening set should greatly enhance the mapping of human disease loci.

DNA Primers↗
PubMed1998

Loss of shaker K channel conductance in 0 K+ solutions: role of the voltage sensor.

In potassium-free solutions some types of K channels enter a long-lasting nonconducting or "defunct" state. It is known that Shaker K channels must open in K+-free solutions to become defunct. Gating current studies presented here indicate an abnormal conformation in the defunct state that restricts S4 movement and alters its kinetics. Thus an abnormality initiated in the P region spreads to the gating apparatus. We find that channels most readily become defunct on repolarization to an intermediate voltage, thus prolonging occupancy of one of the several intermediate closed states. The state dependence of becoming defunct was further dissected by using the gating mutant L382A. Simply closing this channel at 0 mV (reversing the last activation step) does not make the mutant channel defunct. Instead, it is necessary to move further left (more fully closed) in the activation sequence. This was confirmed with ShIR experiments showing that channels become defunct only if there is inward gating charge movement. Rapid transit through the intermediate states, achieved at very negative voltage, is relatively ineffective at making channels defunct. Several mutations that removed C-type inactivation also made the channels resistant to becoming defunct. Our results show that normal gating current cannot be stably recorded in the absence of K+.

Amino Acid Substitution↗
PubMed1998

Charge immobilization caused by modification of internal cysteines in squid Na channels.

We studied the effects of modification of native cysteines present in squid giant axon Na channels with methanethiosulfonates. We find that intracellular, but not extracellular, perfusion of axons with positively charged [(2-trimethylammonium)-ethyl]methanethiosulfonate (MTSET), or 3(triethylammonium)propyl]methanethiosulfonate (MTS-PTrEA) irreversibly reduces sodium ionic (INa) and gating (Ig) currents. The rate of modification of Na channels was dependent on the concentration of the modifying agent and the transmembrane voltage. Hyperpolarized membrane potentials (e.g., -110 mV) protected the channels from modification by MTS-PTrEA. In addition to reducing the amplitudes of INa and Ig, MTS-PTrEA also altered their kinetics such that the remaining INa did not appear to inactivate, whereas Ig was made sharper and declined to baseline more quickly. The shape and amplitude of Ig after modification of channels with MTS-PTrEA appeared to be "charge-immobilized," as if the modified channels were inactivated. MTS-PTrEA did not affect INa or Ig when inactivation was removed by internal perfusion of the axon with pronase. In addition, we find that the steady-state inactivation curve of modified Na channels is made much shallower and is markedly shifted to hyperpolarized potentials. The rates of activation, deactivation, or open-state inactivation were not altered in MTS-PTrEA-modified channels. The uncharged sulfhydryl reagent methymethanethiosulfonate (MMTS) did not affect either INa or Ig, but prevented the irreversible effects of MTS-PTrEA or MTSET on Na channels. It is proposed that the positively charged methanethiosulfonates MTS-PTrEA and MTSET modify a native internal cysteine(s) in squid Na channels, and by doing so promote inactivation from closed states, resulting in charge immobilization and reduction of INa.

Anesthetics, Local↗
PubMed1997

Induction of long-term depression and rebound potentiation by inositol trisphosphate in cerebellar Purkinje neurons.

Cerebellar Purkinje neurons receive two major excitatory inputs, the climbing fibers (CFs) and parallel fibers (PFs). Simultaneous, repeated activation of CFs and PFs results in the long-term depression (LTD) of the amplitude of PF-evoked synaptic currents. To induce LTD, activation of CFs may be substituted with depolarization of the Purkinje neuron to turn on voltage-activated calcium channels and increase the intracellular calcium concentration. The role of PFs in the induction of LTD, however, is less clear. PFs activate glutamate metabotropic receptors that increase phosphoinositide turnover and elevate cytosolic inositol 1,4,5-trisphosphate (InsP3). It has been proposed that calcium release from intracellular stores via InsP3 receptors may be important in the induction of LTD. We studied the role of InsP3 in the induction of LTD by photolytic release of InsP3 from its biologically inactive "caged" precursor in voltage-clamped Purkinje neurons in acutely prepared cerebellar slices. We find that InsP3-evoked calcium release is as effective in LTD induction as activation of PFs. InsP3-induced LTD was prevented by calcium chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N', N'-tetraacetic acid. LTD produced either by repeated activation of PFs combined with depolarization (PF+DeltaV), or by InsP3 combined with depolarization (InsP3+DeltaV) saturated at approximately 50%. Maximal LTD induced by PF+DeltaV could not be further increased by InsP3+DeltaV and vice versa, which suggests that both protocols for induction of LTD share a common path. In addition to inducing LTD, photo-release of InsP3+DeltaV resulted in the rebound potentiation of inhibitory synaptic currents. In the presence of heparin, an InsP3 receptor antagonist, repeated activation of PF+DeltaV failed to induce LTD, suggesting that InsP3 receptors play an important role in LTD induction under physiological conditions.

Animals↗
PubMed1997

Killing K channels with TEA+.

Tetraethylammonium (TEA+) is widely used for reversible blockade of K channels in many preparations. We noticed that intracellular perfusion of voltage-clamped squid giant axons with a solution containing K+ and TEA+ irreversibly decreased the potassium current when there was no K+ outside. Five minutes of perfusion with 20 mM TEA+, followed by removal of TEA+, reduced potassium current to < 5% of its initial value. The irreversible disappearance of K channels with TEA+ could be prevented by addition of > or = 10 mM K+ to the extracellular solution. The rate of disappearance of K channels followed first-order kinetics and was slowed by reducing the concentration of TEA+. Killing is much less evident when an axon is held at -110 mV to tightly close all of the channels. The longer-chain TEA+ derivative decyltriethylammonium (C10+) had irreversible effects similar to TEA+. External K+ also protected K channels against the irreversible action of C10+. It has been reported that removal of all K+ internally and externally (dekalification) can result in the disappearance of K channels, suggesting that binding of K+ within the pore is required to maintain function. Our evidence further suggests that the crucial location for K+ binding is external to the (internal) TEA+ site and that TEA+ prevents refilling of this location by intracellular K+. Thus in the absence of extracellular K+, application of TEA+ (or C10+) has effects resembling dekalification and kills the K channels.

Binding Sites↗
PubMed1997

Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons.

Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.

Animals↗
PubMed1995

Inactivation in ShakerB K+ channels: a test for the number of inactivating particles on each channel.

Fast inactivation in ShakerB K channels results from pore-block caused by "ball peptides" attached to the inner part of each K channel. We have examined the question of how many functional inactivating balls are on each channel and how this number affects inactivation and recovery from inactivation. To that purpose we expressed ShakerB in the insect cell line Sf9 and gradually removed inactivation by perfusing the cell interior with the hydrolytic enzyme papain under whole cell patch clamp. Inactivation slows down as the balls are removed by an amount consistent with the presence of four balls on each channel. Recovery from inactivation has the same time course early and late in papain action; it does not depend on the number of balls remaining on the channel, consistent with the idea that reinactivation is not significant during recovery from inactivation. Our conclusion is that ShakerB has four ball peptides, each capable of causing inactivation. Statistically, the balls are identical and independent. The stability of N-type inactivation by the remaining balls is not appreciably affected by removing some of the balls from a channel.

Animals↗