PubMed Health⌕ Search

Biomedical subjects

C T Bond

Publications and source records attributed to C T Bond.

At least 19 recordsLinked to original sources
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↗
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

An alternative HER-2/neu transcript of 8 kb has an extended 3'UTR and displays increased stability in SKOV-3 ovarian carcinoma cells.

UNLABELLED: HER-2/neu is a potent oncogene that predicts poor outcome when overexpressed in ovarian cancer. The SKOV-3 ovarian carcinoma cell line, one of the only models for HER2-driven ovarian cancer, expresses a major uncharacterized 8-kb alternative HER-2 transcript. OBJECTIVES: The aim of this study was to characterize the structure and determine the origin of the alternative sequence and examine the possible role of the 8-kb alternative transcript in overexpression of the HER-2 gene. METHODS: The structure of the 8-kb transcript was investigated using polymerase chain reaction (PCR) and nucleotide sequencing of cDNA clones. PCR analysis of genomic DNA was used to assess the origin of the 8-kb transcript. The stability of the 8-kb mRNA was assessed by Northern blot analysis of RNA extracted from cells treated with transcriptional inhibitors. RESULTS: Similar 5'UTR and coding sequence but an extended 3'UTR were contained in the 8-kb compared to the well-characterized 4.5-kb HER-2 transcript. Genomic DNA had continuity between the novel 3'UTR sequence from the 8-kb transcript and adjacent HER-2 terminal exon sequence. The 8-kb transcript had a half-life of 13 h compared to 5.5 h for the 4.5-kb transcript (P<0.01). CONCLUSIONS: The 8-kb transcript is generated from alternative polyadenylation site usage rather than gene rearrangement. Since the 8-kb transcript contains alternative sequence found at the 3' end of the normal HER-2 gene, it could be expressed in other cells. Increased stability of the 8-kb transcript may confer a selective advantage for SKOV-3 cells by providing enhanced HER-2 expression.

3' Untranslated Regions↗
PubMed1999

Gene structure and chromosome mapping of the human small-conductance calcium-activated potassium channel SK1 gene (KCNN1).

Small-conductance, calcium-activated potassium channels contribute to the afterhyperpolarization in central neurons and other cell types. Because these channels regulate neuronal excitability, defects in their genes could cause excitability disorders. The human cDNA encoding one such channel, SK1 (KCNN1), was recently cloned. Here we describe the gene structure of KCNN1 and its localization by radiation hybrid mapping to chromosome 19p13.1.

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↗
PubMed1997

A human intermediate conductance calcium-activated potassium channel.

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is approximately 50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 microM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3. 5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 microM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Amino Acid Sequence↗
PubMed1996

Small-conductance, calcium-activated potassium channels from mammalian brain.

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Amino Acid Sequence↗
PubMed1996

Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co-injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co-expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4-5 or 4-5-4-5, but not when they were connected 4-4-5-5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4-5-4-5. Further, the relative order of subunits within the channel contributes to their functional properties.

Animals↗
PubMed1996

Inhibitory interactions between two inward rectifier K+ channel subunits mediated by the transmembrane domains.

Inwardly rectifying K+ channel subunits may form homomeric or heteromeric channels with distinct functional properties. Hyperpolarizing commands delivered to Xenopus oocytes expressing homomeric Kir 4.1 channels evoke inwardly rectifying K+ currents which activate rapidly and undergo a pronounced decay at more hyperpolarized potentials. In addition, Kir 4.1 subunits form heteromeric channels when coexpressed with several other inward rectifier subunits. However, coexpression of Kir 4.1 with Kir 3.4 causes an inhibition of the Kir 4.1 current. We have investigated this inhibitory effect and show that it is mediated by interactions between the predicted transmembrane domains of the two subunit classes. Other subunits within the Kir 3.0 family also exhibit this inhibitory effect which can be used to define subgroups of the inward rectifier family. Further, the mechanism of inhibition is likely due to the formation of an "inviable complex" which becomes degraded, rather than by formation of stable nonconductive heteromeric channels. These results provide insight into the assembly and regulation of inwardly rectifying K+ channels and the domains which define their interactions.

Animals↗
PubMed1996

Characterization of two new preproGnRH mRNAs in the tree shrew: first direct evidence for mesencephalic GnRH gene expression in a placental mammal.

Reproductive maturation and regulation is centrally orchestrated by gonadotropin-releasing hormone (GnRH). GnRH produced in the vertebrate hypothalamus acts on the pituitary to regulate gonadotropins. In nonplacental mammalian species, it has recently been shown that a second GnRH gene is expressed in mesencephalic cells. Here, we report the cDNA sequences and expression patterns for two distinct genes encoding the hypothalamic and mesencephalic GnRH forms in the brain of a placental mammal, the tree shrew (Tupaia glis belangeri). The novel mammalian GnRH form, designated here as [His5Trp7Tyr8]GnRH (often called chicken GnRH II), is expressed in neurons of the mesencephalon and is the first nonhypothalamic form to be isolated from a mammal. Its peptide sequence is identical to the form previously reported in fish, amphibians, reptiles, and birds, revealing that it has remained unchanged for 500 million years. In contrast, the sequences of the hypothalamic GnRH decapeptides vary by as much as 50% across vertebrate species. The remarkable sequence conservation of mesencephalic GnRH suggests that it has been highly constrained throughout evolution, perhaps indicating an important, conserved nongonadotropic role. The discovery and localization of two mRNAs encoding distinct GnRH forms in an advanced mammal suggest that other mammals, including primates, may also have a second GnRH gene with expression localized in the midbrain.

Amino Acid Sequence↗
PubMed1996

Inward rectifier potassium channels. Cloning, expression and structure-function studies.

A PCR-based cloning strategy was used to identify novel subunits of the two-transmembrane domain inward rectifier potassium channel family from rat brain, heart, and skeletal muscle. When expressed in Xenopus oocytes, two of these clones (Kir4.1 and Kir2.3) gave rise to inwardly rectifying potassium currents. Two-electrode voltage clamp commands to potentials negative to EK evoked inward potassium-selective currents which rapidly reached a peak amplitude and then relaxed to a steady-state level. Differences in the extent of current relaxation, the degree of rectification, and the voltage-dependent block by external cesium were detected. Two other members of this family (Kir5.1 and Kir3.4) did not produce macroscopic currents, when expressed by themselves, yet both subunits modified the currents when coexpressed with other specific members of the Kir family. Expression of chimeric subunits between Kir4.1 and either Kir5.1 or Kir3.4 suggested that the transmembrane domains determine the specificity of subunit heteropolymerization, while the C-terminal domains contribute to alterations in activation kinetics and rectification. Expression of covalently linked subunits demonstrated that the relative subunit positions, as well as stoichiometry, affect heteromeric channel activity.

Animals↗
PubMed1995

Three gonadotropin-releasing hormone genes in one organism suggest novel roles for an ancient peptide.

Gonadotropin-releasing hormone (GnRH) is known and named for its essential role in vertebrate reproduction. Release of this decapeptide from neurons in the hypothalamus controls pituitary gonadotropin levels which, in turn, regulate gonadal state. The importance of GnRH is underscored by its widespread expression and conservation across vertebrate taxa: five amino acids are invariant in all nine known forms, whereas two others show only conservative changes. In most eutherian mammals, only one form, expressed in the hypothalamus, is thought to exist, although in a recent report, antibody staining in developing primates suggests an additional form. In contrast, multiple GnRH forms and expression loci have been reported in many non-mammalian vertebrates. However, evidence based on immunological discrimination does not always agree with analysis of gene expression, since GnRH forms encoded by different genes may not be reliably distinguished by antibodies. Here we report the expression of three distinct GnRH genes in a teleost fish brain, including the sequence encoding a novel GnRH preprohormone. Using in situ hybridization, we show that this form is found only in neurons that project to the pituitary and exhibit changes in soma size depending on social and reproductive state. The other two GnRH genes are expressed in other, distinct cell populations. All three genes share the motif of encoding a polypeptide consisting of GnRH and a GnRH-associated peptide. Whereas the GnRH moiety is highly conserved, the GnRH-associated peptides are not, reflecting differential selective pressure on different parts of the gene. GnRH forms expressed in nonhypothalamic regions may serve to coordinate reproductive activities of the animal.

Amino Acid Sequence↗
PubMed1995

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic beta-cells and in the brain.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Amino Acid Sequence↗
PubMed1995

Episodic ataxia results from voltage-dependent potassium channels with altered functions.

Episodic ataxia (EA) is an autosomal dominant human disorder that produces persistent myokymia and attacks of generalized ataxia. Recently, familial EA has been linked to the voltage-dependent delayed rectifier, Kv1.1, on chromosome 12. Six EA families have been identified that carry distinct Kv1.1 missense mutations; all individuals are heterozygous. Expression in Xenopus oocytes demonstrates that two of the EA subunits form homomeric channels with altered gating properties. V408A channels have voltage dependence similar to that of wild-type channels, but with faster kinetics and increased C-type inactivation, while the voltage dependence of F184C channels is shifted 20 mV positive. The other four EA subunits do not produce functional homomeric channels but reduce the potassium current when coassembled with wild-type subunits. The results suggest a cellular mechanism underlying EA in which the affected nerve cells cannot efficiently repolarize following an action potential because of altered delayed rectifier function.

Animals↗