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D M Papazian

Publications and source records attributed to D M Papazian.

35 records · Page 2Linked to original sources
PubMed1994

Glycosylation of shaker potassium channel protein in insect cell culture and in Xenopus oocytes.

We have studied the glycosylation of Shaker K+ channel protein made in two expression systems: an insect cell culture line and amphibian oocytes. In both systems, two potential sites for N-linked glycosylation were modified. The modified sites were located between the first and second putative transmembrane segments, S1 and S2. Although the same sites appeared to be glycosylated in both systems, the fraction of protein glycosylated and the size, structure, or composition of the oligosaccharide chains added were quite different. The results indicate that the S1-S2 loop is extracellular, consistent with a cytoplasmic location for the N-terminus and a transmembrane disposition for hydrophobic segment S1. We have also shown that glycosylation occurs in two stages in oocytes, generating an immature and a mature form of Shaker protein. However, glycosylation is not required either for the assembly of functional channels or for their transport to the cell surface.

Amino Acid Sequence↗
PubMed1994

Purification and reconstitution of functional Shaker K+ channels assayed with a light-driven voltage-control system.

Voltage-dependent potassium channels are integral membrane proteins that control the excitability of nerve and muscle. The cloning of genes for K+ channels has led to structure/function analysis using a combination of site-directed mutagenesis and electrophysiology. As a result, much has been learned about how these proteins work. A deeper understanding of their function will require detailed structural characterization, however. We now report the purification of Shaker K+ channels from an insect expression system using immunoaffinity methods. The purified channels have been reconstituted, assayed using a novel, light-driven, vesicular voltage-control system, and shown to be functional. This approach will enable us to compare and optimize methods for protein production and purification. Purification of active protein is a prerequisite for detailed structural analysis, since activity is the key indication that the structural integrity of the channel has been preserved during biochemical procedures. Thus, this work represents a first step toward the determination of the structure of Shaker K+ channels.

Amino Acid Sequence↗
PubMed1994

S4 mutations alter gating currents of Shaker K channels.

Activation of voltage-dependent channels involves charge-moving conformational changes of the voltage sensor that can be detected as gating currents. In Shaker K channels, the S4 sequence comprises at least part of the voltage sensor. We have measured gating currents in three S4 mutants: R368Q, R377K, and R371Q. R368Q enhances the separation of two components of charge movement and greatly reduces the valence of one component. R377K partially uncouples charge movement from channel opening. In contrast, the gating currents of R371Q resemble those of the control. Two other S4 mutations, R377Q and K374Q, make proteins that are not properly processed and transported to the cell surface and thereby eliminate the gating current. To explain the effects of R368Q, we hypothesize that R368 is part of a salt bridge that is broken early in activation. Subsequently, the S4 segment undergoes a conformational change, and, after a final, relatively voltage-independent step, the channel opens.

Amino Acid Sequence↗
PubMed1993

S4 mutations alter the single-channel gating kinetics of Shaker K+ channels.

The S4 sequence comprises at least part of the voltage sensor in Shaker K+ channels. Two site-directed mutations in the Shaker S4 sequence, R368Q and R377K, which decrease the voltage dependence of the whole-cell current, alter voltage-dependent gating at the single-channel level. Compared with the wild-type channel, they increase the latency to first opening, destabilize the open state, and alter the equilibria of voltage-dependent transitions, so that some of the charge movement occurs after the first opening. Whether these changes reflect a new mechanism of activation is a key question. Although the mutations alter the kinetics of many steps in the gating process, we conclude that the mutant channels are likely to activate using the same fundamental mechanism as wild-type channels.

Animals↗
PubMed1992

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.

Animals↗
PubMed1991

Molecular basis of gating charge immobilization in Shaker potassium channels.

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.

Amino Acid Sequence↗
PubMed1991

Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence.

Voltage-dependent potassium, sodium and calcium ion channels may share a common mechanism of activation, in which the conserved S4 sequence acts as the primary voltage sensor. Site-directed mutagenesis of the S4 sequence of the Shaker potassium channel and electrophysiological analysis suggest that voltage-dependent activation involves the S4 sequence but is not solely due to electrostatic interactions.

Animals↗
PubMed1990

Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line.

We constructed a recombinant baculovirus, A. californica nuclear polyhedrosis virus, containing the Drosophila Shaker H4 K+ channel cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the cell line Sf9, derived from the army-worm caterpillar S. frugiperda, expresses fully functional Shaker transient K+ currents, as assayed by whole-cell recording. K+ currents begin to appear at about 15 hr after infection, and they continue to increase over the next 3 days. Over the same period of time, a 75 kd band appears on SDS gels stained with Coomassie blue. The identity of this band as a Shaker gene product is confirmed by Western blot analysis using an anti-Shaker antiserum. The 75 kd band accounts for a substantial fraction of the membrane protein in Shaker-infected Sf9 cells. These results give hope that the baculovirus system, which has been used successfully for high-level expression of soluble proteins from higher eukaryotes, may be appropriate for producing large amounts of cloned ion channel proteins as well.

Animals↗
PubMed1990

Immunological characterization of K+ channel components from the Shaker locus and differential distribution of splicing variants in Drosophila.

Antibodies were raised to three portions of the predicted sequences of Shaker, a gene that encodes a family of K+ channel components that are produced by the alternative splicing of transcripts. On immunoblots, the protein products appear to be 65,000-85,000 daltons in size. No smaller products were detected. Immunocytochemistry has revealed a nonuniform distribution of Shaker products in the brain of the adult fly. By comparing antisera directed against regions shared by all the splicing variants to antisera that are directed against one particular group of splicing variants, we have determined that there is a differential distribution of that group of variants. Thus, the alternative splicing of Shaker transcripts appears to produce different subtypes of A-channels in different tissues.

Amino Acid Sequence↗
PubMed1988

Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes.

The Shaker gene of Drosophila melanogaster encodes a potassium-selective ion channel, the 'A' channel, or one of its subunits. A single Shaker messenger RNA species suffices to direct the synthesis of functional A channels in Xenopus oocytes. Physiological characteristics of the A currents induced by two different mRNA species are compared.

Animals↗
PubMed1987

Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.

On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.

Animals↗
PubMed1987

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.

Animals↗
PubMed1984

Partial purification and functional identification of a calmodulin-activated, adenosine 5'-triphosphate-dependent calcium pump from synaptic plasma membranes.

Synaptic plasma membranes isolated from rat brain contain a calmodulin-activated Ca2+ pump. It has been purified 80- to 160-fold by solubilization with Triton X-100 and affinity chromatography on a calmodulin-Sepharose 4B column. After reconstitution into phospholipid vesicles, the affinity-purified pump efficiently catalyzed ATP dependent Ca2+ transport, which was activated 7- to 9-fold by calmodulin. The major protein component of the affinity-purified preparation had a Mr = 140,000; it was virtually the only band visualized on a Coomassie blue-stained SDS polyacrylamide gel. It has been identified as the Ca2+ pump by two functional criteria. First, it was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner; the phosphorylated protein had the chemical reactivity of an acyl phosphate, characteristic of the phosphorylated intermediates of ion-transporting ATPases. Second, the protein was enriched by transport-specific fractionation, a density gradient procedure which uses the transport properties of the reconstituted Ca2+ pump as a physical tool for its purification. By analogy with calmodulin-activated (Ca2+ + Mg2+) ATPases of other cell types, and because of its presence in a synaptic plasma membrane fraction, we hypothesize that the calmodulin-activated Ca2+ pump functions in vivo to extrude Ca2+ from nerve terminals.

Adenosine Triphosphate↗