A family of three mouse potassium channel genes with intronless coding regions

To understand the molecular mechanisms responsible for generating physiologically diverse potassium channels in mammalian cells, mouse genomic clones have been isolated with a potassium channel complementary DNA, MBK1, that is homologous to the Drosophila potassium channel gene, Shaker. A family of three closely related potassium channel genes (MK1, MK2, and MK3) that are encoded at distinct genomic loci has been isolated. Sequence analysis reveals that the coding region of each of these three genes exists as a single uninterrupted exon in the mouse genome. This organization precludes the generation of multiple forms of the protein by alternative RNA splicing, a mechanism known to characterize the Drosophila potassium channel genes Shaker and Shab. Thus, mammals may use a different strategy for generating diverse K+ channels by encoding related genes at multiple distinct genomic loci, each of which produces only a single protein.     

A Drosophila mechanosensory transduction channel

Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, we analyzed Drosophila melanogaster mechanoreceptive mutants for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolished mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs.     

Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila

Potassium currents are crucial for the repolarization of electrically excitable membranes, a role that makes potassium channels a target for physiological modifications that alter synaptic efficacy. The Shaker locus of Drosophila is thought to encode a K+ channel. The sequence of two complementary DNA clones from the Shaker locus is reported here. The sequence predicts an integral membrane protein of 70,200 daltons containing seven potential membrane-spanning sequences. In addition, the predicted protein is homologous to the vertebrate sodium channel in a region previously proposed to be involved in the voltage-dependent activation of the Na+ channel. These results support the hypothesis that Shaker encodes a structural component of a voltage-dependent K+ channel and suggest a conserved mechanism for voltage activation.     

New genes in Drosophila quickly become essential

To investigate the origin and evolution of essential genes, we identified and phenotyped 195 young protein-coding genes, which originated 3 to 35 million years ago in Drosophila. Knocking down expression with RNA interference showed that 30% of newly arisen genes are essential for viability. The proportion of genes that are essential is similar in every evolutionary age group that we examined. Under constitutive silencing of these young essential genes, lethality was high in the pupal stage and also found in the larval stages. Lethality was attributed to diverse cellular and developmental defects, such as organ formation and patterning defects. These data suggest that new genes frequently and rapidly evolve essential functions and participate in development.     

A component of calcium-activated potassium channels encoded by the Drosophila slo locus.

Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.     

Transient expression of homologous genes in Drosophila cells

A cloned Drosophila heat shock protein 22 gene was transfected into two independently established Drosophila cell lines. Each line carried a different heat shock protein 22 allele, distinguishable by electrophoresis of the protein. The transfected gene was not expressed at 25 degrees C but could be induced at 36 degrees C. In one line, two heat shock protein 22 electromorphs were synthesized.     

A mutation in the C. elegans EXP-2 potassium channel that alters feeding behavior

The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go-related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.     

Expression of a cloned rat brain potassium channel in Xenopus oocytes

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.     

Increasing the multiplicity of ribosomal RNA genes in Drosophila melanogaster

In wild-type Drosophila melanogaster females there are about 250 ribosomal RNA genes in each nucleolus organizer region of the two X chromosomes. When this same nucleolus organizer region is present in flies in only a single dose, the number of ribosomal RNA genes increases to approximately 400. This increase is most easily explained by disproportionate replication of these genes.     

Single-channel and genetic analyses reveal two distinct A-type potassium channels in Drosophila

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the channels underlying the fast transient potassium current (A current) in cultured myotubes and neurons of Drosophila. The myotube (A1) and neuronal (A2) channels are distinct, differing in conductance, voltage dependence, and gating kinetics. The myotube currents have a faster and more voltage-dependent macroscopic inactivation rate, a larger steady-state component, and a less negative steady-state inactivation curve than the neuronal currents. The myotube channels have a conductance of 12 to 16 picosiemens, whereas the neuronal channels have a conductance of 5 to 8 picosiemens. In addition, the myotube channel is affected by Shaker mutations, whereas the neuronal channel is not. Together, these data suggest that the two channels are separate molecular structures, the expression of which is controlled, at least in part, by different genes.     

External calcium ions are required for potassium channel gating in squid neurons

The effects of calcium removal on the voltage-dependent potassium channels of isolated squid neurons were studied with whole cell patch-clamp techniques. When the calcium ion concentration was lowered from 10 to 0 millimolar (that is, no added calcium), potassium channel activity, identified from its characteristic time course, disappeared within a few seconds and there was a parallel increase in resting membrane conductance and in the holding current. The close temporal correlation of the changes in the three parameters suggests that potassium channels lose their ability to close in the absence of calcium and simultaneously lose their selectivity. If potassium channels were blocked by barium ion before calcium ion was removed, the increases in membrane conductance and holding current were delayed or prevented. Thus calcium is an essential cofactor in the gating of potassium channels in squid neurons.     

Graded regulation of the Kv2.1 potassium channel by variable phosphorylation

Dynamic modulation of ion channels by phosphorylation underlies neuronal plasticity. The Kv2.1 potassium channel is highly phosphorylated in resting mammalian neurons. Activity-dependent Kv2.1 dephosphorylation by calcineurin induces graded hyperpolarizing shifts in voltage-dependent activation, causing suppression of neuronal excitability. Mass spectrometry-SILAC (stable isotope labeling with amino acids in cell culture) identified 16 Kv2.1 phosphorylation sites, of which 7 were dephosphorylated by calcineurin. Mutation of individual calcineurin-regulated sites to alanine produced incremental shifts mimicking dephosphorylation, whereas mutation to aspartate yielded equivalent resistance to calcineurin. Mutations at multiple sites were additive, showing that variable phosphorylation of Kv2.1 at a large number of sites allows graded activity-dependent regulation of channel gating and neuronal firing properties.     

Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel

Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.     

Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. Here we show that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. Our results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species.     

Identification of a family of muscarinic acetylcholine receptor genes

Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.     

Crystal structure of the human two-pore domain potassium channel K2P1

Two-pore domain potassium (K(+)) channels (K2P channels) control the negative resting potential of eukaryotic cells and regulate cell excitability by conducting K(+) ions across the plasma membrane. Here, we present the 3.4 angstrom resolution crystal structure of a human K2P channel, K2P1 (TWIK-1). Unlike other K(+) channel structures, K2P1 is dimeric. An extracellular cap domain located above the selectivity filter forms an ion pathway in which K(+) ions flow through side portals. Openings within the transmembrane region expose the pore to the lipid bilayer and are filled with electron density attributable to alkyl chains. An interfacial helix appears structurally poised to affect gating. The structure lays a foundation to further investigate how K2P channels are regulated by diverse stimuli.     

Protein kinase activity closely associated with a reconstituted calcium-activated potassium channel.

Modulation of the activity of potassium and other ion channels is an essential feature of nervous system function. The open probability of a large conductance Ca(2+)-activated K+ channel from rat brain, incorporated into planar lipid bilayers, is increased by the addition of adenosine triphosphate (ATP) to the cytoplasmic side of the channel. This modulation takes place without the addition of protein kinase, requires Mg2+, and is mimicked by an ATP analog that serves as a substrate for protein kinases but not by a nonhydrolyzable ATP analog. Addition of protein phosphatase 1 reverses the modulation by MgATP. Thus, there may be an endogenous protein kinase activity firmly associated with this K+ channel. Some ion channels may exist in a complex that contains regulatory protein kinases and phosphatases.     

Crystal structure of a mammalian voltage-dependent Shaker family K+ channel

Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.