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J Maylie

Publications and source records attributed to J Maylie.

At least 19 recordsLinked to original sources
PubMed2001

Small-conductance calcium-activated potassium currents in mouse hyperexcitable denervated skeletal muscle.

1. Hyperexcitability in denervated skeletal muscle is associated with the expression of SK3, a small-conductance Ca2+-activated K+ channel (SK channel). SK currents were examined in dissociated fibres from flexor digitorum brevis (FDB) muscle using the whole-cell patch clamp configuration. 2. Depolarization activated a K+-selective, apamin-sensitive and iberiotoxin-insensitive current, detected as a tail current upon repolarization, in fibres from denervated but not innervated muscle. Dialysis of the fibres with 20 mM EGTA in the patch pipette solution eliminated the tail current, consistent with this current reflecting Ca2+-activated SK channels expressed only in denervated muscle. 3. Activation of SK tail currents depended on the duration of the depolarizing pulse, consistent with a rise in intracellular Ca2+ due to release from the sarcoplasmic reticulum (SR) and influx through voltage-gated Ca2+ channels. 4. The envelope of SK tail currents was diminished by 10 microM ryanodine for all pulse durations, whereas 2 mM cobalt reduced the SK tail current for pulses greater than 80 ms, demonstrating that Ca2+ release from the SR during short pulses primarily activated SK channels. 5. In current clamp mode with the resting membrane potential set at -70 mV, denervation decreased the action potential threshold by approximately 8 mV. Application of apamin increased the action potential threshold in denervated fibres to that measured in innervated fibres, suggesting that SK channel activity modulates the apparent action potential threshold. 6. These results are consistent with a model in which SK channel activity in the T-tubules of denervated skeletal muscle causes a local increase in K+ concentration that results in hyperexcitability.

Action Potentials↗
PubMed2001

Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1.

Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

3' Untranslated Regions↗
PubMed2000

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

5' Untranslated Regions↗
PubMed2000

Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy.

Expansion of a CTG trinucleotide repeat in the 3' UTR of the gene DMPK at the DM1 locus on chromosome 19 causes myotonic dystrophy, a dominantly inherited disease characterized by skeletal muscle dystrophy and myotonia, cataracts and cardiac conduction defects. Targeted deletion of Dm15, the mouse orthologue of human DMPK, produced mice with a mild myopathy and cardiac conduction abnormalities, but without other features of myotonic dystrophy, such as myotonia and cataracts. We, and others, have demonstrated that repeat expansion decreases expression of the adjacent gene SIX5 (refs 7,8), which encodes a homeodomain transcription factor. To determine whether SIX5 deficiency contributes to the myotonic dystrophy phenotype, we disrupted mouse Six5 by replacing the first exon with a beta-galactosidase reporter. Six5-mutant mice showed reporter expression in multiple tissues, including the developing lens. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Our results suggest that SIX5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.

3' Untranslated Regions↗
PubMed2000

Right ventricular systolic pressure load alters myocyte maturation in fetal sheep.

The effects of right ventricular (RV) systolic pressure (RVSP) load on fetal myocyte size and maturation were studied. Pulmonary artery (PA) pressure was increased by PA occlusion from mean 47.4 +/- 5.0 (+/-SD) to 71 +/- 13.6 mmHg (P < 0.0001) in eight RVSP-loaded near-term fetal sheep for 10 days. The maximal pressure generated by the RV with acute PA occlusion increased after RVSP load: 78 +/- 7 to 101 +/- 15 mmHg (P < 0.005). RVSP-load hearts were heavier (44.7 +/- 8.4 g) than five nonloaded hearts (31.8 +/- 0.2 g; P < 0.03); heart-to-body weight ratio (10.9 +/- 1.1 and 6.5 +/- 0.9 g/kg, respectively; P < 0.0001). RVSP-RV myocytes were longer (101.3 +/- 10.2 microm) than nonloaded RV myocytes (88.2 +/- 8.1 microm; P < 0. 02) and were more often binucleated (82 +/- 13%) than nonloaded myocytes (63 +/- 7%; P < 0.02). RVSP-loaded myocytes had less myofibrillar volume than did nonloaded hearts (44.1 +/- 4.4% and 56. 1 +/- 2.6%; P < 0.002). We conclude that RV systolic load 1) leads to RV myocyte enlargement, 2) has minor effects on left ventricular myocyte size, and 3) stimulates maturation (increased RV myocyte binucleation). Myocyte volume data suggest that RV systolic loading stimulates both hyperplastic and hypertrophic growth.

Animals↗
PubMed1999

Domains responsible for constitutive and Ca(2+)-dependent interactions between calmodulin and small conductance Ca(2+)-activated potassium channels.

Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca(2+)-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca(2+)-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca(2+)-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca(2+)-dependent and Ca(2+)-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

Animals↗
PubMed1999

Small-conductance calcium-activated potassium channels.

SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetitive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin-sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin-insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin-sensitive and apamin-insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

Action Potentials↗
PubMed1999

Skeletal muscle and small-conductance calcium-activated potassium channels.

Skeletal muscle becomes hyperexcitable following denervation and when cultured in the absence of nerve cells. In these circumstances, the bee venom peptide toxin apamin, a blocker of small-conductance calcium-activated potassium (SK) channels, dramatically reduces the hyperexcitability. In this report, we show that SK3 channels are expressed in denervated skeletal muscle and in L6 cells. Action potentials evoked from normal innervated rat skeletal muscle did not exhibit an afterhyperpolarization, indicating a lack of SK channel activity; very low levels of apamin binding sites, SK3 protein, or SK3 mRNA were present. However, denervation resulted in apamin-sensitive afterhyperpolarizations and increased apamin binding sites, SK3 protein, and SK3 mRNA. Cultured rat L6 myoblasts and differentiated L6 myotubes contained similar levels of SK3 mRNA, although apamin-sensitive SK currents and apamin binding sites were detected only following myotube differentiation. Therefore, different molecular mechanisms govern SK3 expression levels in denervated muscle compared with muscle cells differentiated in culture.

Animals↗
PubMed1999

Bicuculline block of small-conductance calcium-activated potassium channels.

Small-conductance calcium-activated potassium channels (SK channels) are gated solely by intracellular calcium ions and their activity is responsible for the slow afterhyperpolarization (AHP) that follows an action potential in many excitable cells. Brain slice studies commonly employ a methyl derivative of bicuculline (bicuculline-m), a GABAA (gamma-aminobutyric acid) receptor antagonist, to diminish the tonic inhibitory influences of GABAergic synapses, or to investigate the role of these synapses in specialized neural networks. However, recent evidence suggests that bicuculline-m may not be specific for GABAA receptors and may also block the slow AHP. Therefore, the effects of bicuculline-m on cloned apamin-sensitive SK2 and apamin-insensitive SK1 channels were examined following expression in Xenopus oocytes. The results show that at concentrations employed for slice recordings, bicuculline-m potently blocks both apamin-sensitive SK2 currents and apamin-insensitive SK1 currents when applied to outside-out patches. Apamin-insensitive SK1 currents run down in excised patches. The potency of bicuculline-m block also decreases with time after patch excision. Site-directed mutagenesis that changes two residues in the outer vestibule of the SK1 pore that confers apamin sensitivity also reduces run down of the current in patches, and endows stable sensitivity to bicuculline-m indistinguishable from SK2. Therefore, the use of bicuculline-m in slice recordings may mask apamin-sensitive slow AHPs that are important determinants of neuronal excitability. In addition, bicuculline-m-insensitive slow AHPs may indicate that the underlying channels have run down.

Animals↗
PubMed1999

Gating properties of single SK channels in hippocampal CA1 pyramidal neurons.

The activation of small-conductance calcium-activated potassium channels (SK) has a profound effect on membrane excitability. In hippocampal pyramidal neurons, SK channel activation by Ca2+ entry from a preceding burst of action potentials generates the slow afterhyperpolarization (AHP). Stimulation of a number of receptor types suppresses the slow AHP, inhibiting spike frequency adaptation and causing these neurons to fire tonically. Little is known of the gating properties of native SK channels in CNS neurons. By using excised inside-out patches, a small-amplitude channel has been resolved that was half-activated by approximately 0.6 microM Ca2+ in a voltage-independent manner. The channel possessed a slope conductance of 10 pS and exhibited nonstationary gating. These properties are in accord with those of cloned SK channels. The measured Ca2+ sensitivity of hippocampal SK channels suggests that the slow AHP is generated by activation of SK channels from a local rise of intracellular Ca2+.

Animals↗
PubMed1998

Mechanism of calcium gating in small-conductance calcium-activated potassium channels.

The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

Animals↗
PubMed1998

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Animals↗
PubMed1998

Characterization of three episodic ataxia mutations in the human Kv1.1 potassium channel.

Episodic ataxia (EA) is a rare inherited neurological disorder due to mutation in the voltage-dependent Kv1.1 potassium channel. In nine unrelated families, a different missense point mutation at highly conserved positions has been reported. We have previously characterized six of the EA mutants. In this study, three recently identified mutations were introduced into the human Kv1.1 cDNA and expressed in Xenopus oocytes. Compared to wild type, T226A and T226M reduced the current amplitude by > 95%, shifted the voltage dependence by 15 mV, and slowed activation and deactivation kinetics. Currents from G311S were approximately 25% of wild type, less steeply voltage-dependent and had more pronounced C-type inactivation. These altered gating properties will reduce the delayed-rectifier potassium current which may underlie the symptoms of EA.

Animals↗
PubMed1998

Episodic ataxia mutations in Kv1.1 alter potassium channel function by dominant negative effects or haploinsufficiency.

Subunits of the voltage-gated potassium channel Kv1.1 containing mutations responsible for episodic ataxia (EA), a human inherited neurological disease, were expressed in Xenopus oocytes. Five EA subunits formed functional homomeric channels with lower current amplitudes and altered gating properties compared with wild type. Two EA mutations located in the first cytoplasmic loop, R239S and F249I, yielded minimal or no detectable current, and Western blot analysis showed reduced protein levels. Coinjection of equal amounts of EA and wild-type mRNAs, mimicking the heterozygous condition, resulted in current amplitudes and gating properties that were intermediate between wild-type and EA homomeric channels, suggesting that heteromeric channels are formed with a mixed stoichiometry of EA and wild-type subunits. To examine the relative contribution of EA subunits in forming heteromeric EA and wild-type channels, each EA subunit was made insensitive to TEA, TEA-tagged, and coexpressed with wild-type subunits. TEA-tagged R239S and F249I induced the smallest shift in TEA sensitivity compared with homomeric wild-type channels, whereas the other TEA-tagged EA subunits yielded TEA sensitivities similar to coexpression of wild-type and TEA-tagged wild-type subunits. Taken together, these results show that the different mutations in Kv1.1 affect channel function and indicate that both dominant negative effects and haplotype insufficiency may result in the symptoms of EA.

Animals↗
PubMed1998

Episodic ataxia type-1 mutations in the hKv1.1 cytoplasmic pore region alter the gating properties of the channel.

Episodic ataxia type-1 is a rare human neurological syndrome which occurs during childhood and persists through the whole life of affected patients. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene hKv1.1 of different affected families. V408A and E325D mutations are located in the cytoplasmic putative pore region of hKv1.1 channels and profoundly alter their gating properties. V408A channels showed increased kinetic rates of activation, deactivation and C-type inactivation. Expression of E325D channels in Xenopus oocytes led to an approximately 13-fold current amplitude reduction and to a 52.4 mV positive shift in the voltage dependence of activation. Moreover, the E325D mutation altered the kinetics of activation, deactivation, C-type inactivation and channel open probability. Heteromeric channels composed of two wild-type and two mutated subunits, linked as dimers, showed gating properties intermediate between channels formed from four normal or four mutated subunits. The results demonstrate that the highly conserved residues Val408 and Glu325 play a pivotal role in several gating processes of a human potassium channel, and suggest a pathogenetic mechanism by which the impairment of the delayed-rectifier function of affected neurons is related to the type and number of mutated subunits which make up the hKv1.1 channels.

Animals↗
PubMed1998

Gating of I(sK) channels expressed in Xenopus oocytes.

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+ current, I(Ks), in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopus oocytes results in I(Ks)-like currents, I(sK), due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of I(sK) suggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40 mV from holding potentials between -60 and -120 mV showed an unusual "cross-over," with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100 ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac I(Ks) may suppress premature action potentials.

Animals↗