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L Salkoff

Publications and source records attributed to L Salkoff.

At least 37 records · Page 2Linked to original sources
PubMed1995

Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels.

Insulin secretion is associated with changes in pancreatic beta-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified beta-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels.

Amino Acid Sequence↗
PubMed1995

A human calcium-activated potassium channel gene expressed in vascular smooth muscle.

Large-conductance Ca(2+)-activated K+ (BK) channels are widespread and functionally heterogeneous. In other classes of K+ channels, functional heterogeneity derives from large gene families, alternative splicing, heterologous subunit composition, and functional modulation. The molecular basis of mammalian BK channel heterogeneity is unknown, since only a single gene (mSlo) has been identified. BK channels in native vascular smooth muscle have an apparent Ca2+ sensitivity approximately 10-fold greater than native brain or skeletal muscle channels, or cloned mSlo channels. Using mSlo as a low-stringency probe, we screened human arterial smooth muscle and genomic libraries extensively in search of genes or splice variants with novel properties. We isolated the human homologue of mSlo, including two novel splice variant forms, but found no other related genes. Electrophysiological characterization of the hSlo clones in Xenopus oocytes and Chinese hamster ovary cells gave BK currents that were not measurably different from mSlo currents. However, coexpression of hSlo with a recently cloned beta-subunit derived from smooth muscle dramatically increased apparent Ca2+ sensitivity. Thus alpha-subunits alone may not determine Ca2+ sensitivity of vascular smooth muscle BK channels. hSlo was mapped to human chromosome 10q23.1, and the genomic structure was analyzed. Immediately after the amino terminal, two unusual regions of trinucleotide repeating sequences are present. The first of these regions encodes polyglycine, and the second encodes polyserine. Both regions of repeated sequence are conserved between the mouse and human genome.

Amino Acid Sequence↗
PubMed1995

The major delayed rectifier in both Drosophila neurons and muscle is encoded by Shab.

The delayed rectifier K+ current in Drosophila is similar to the classical delayed rectifier, originally described by Hodgkin and Huxley. Drosophila provides unique tools of mutant analysis to unambiguously determine the genetic identity of this native K+ current. We identified the Shab gene as the exclusive gene underlying delayed rectifier currents in both muscle and neurons. In muscles, a genetic mutation of Shab removes virtually all the whole cell delayed rectifier current (IK), while leaving unaltered the transient A-current encoded by the Shaker gene. In neurons, the Shab mutation also removes the bulk of IK, but leaves unaltered the transient A-current encoded by the Shal gene. Although most of the delayed rectifier current is the product of the Shab gene, the Shaw gene contributes a small "leak" current to most neurons and muscle cells. Thus, in contrast to the A-currents which are encoded by different genes in muscle and neuronal cell bodies (Shaker and Shal, respectively), the predominant IK in both muscle and neurons is encoded by the same gene (Shab). With the genetic identity of IK confirmed, all of the major K+ currents in embryonic Drosophila neurons and muscle are now known.

Animals↗
PubMed1995

Genetic analysis of Drosophila neurons: Shal, Shaw, and Shab encode most embryonic potassium currents.

In this study, we perform the first genetic analysis of K+ currents in Drosophila embryonic neurons revealing the identity of the currents present. Unlike muscles, where the presence of Shaker is obvious, Shaker currents are not detectable in these neurons. In contrast, we show that Shal is as important in these neuronal cell bodies as Shaker is in muscles. Only three single-channel currents were found, all of which are genetically separable. Shal encodes a 4 pS transient channel. Whole-cell Shal currents have a wide variety of inactivation rates which, in contrast to a mechanism such as heteromultimer formation, is due to single Shal channels assuming different gating modes. Shaw encodes a 42 pS noninactivating channel distinctive for its extremely low voltage sensitivity; Shaw channels have a total equivalent gating charge of 0.90 e- charges, in sharp contrast to 7 e- reported for Shaker channels. An 11 pS slowly inactivating channel also present in these neurons may be encoded by the Shab gene. Thus, of four voltage-dependent K+ channel genes now cloned in Drosophila, all except Shaker appear to be expressed in the cell bodies of these neurons. It is only in Drosophila that a study such as this one can be done. Because Drosophila contains only a single member of each of the four subfamilies of K+ channel genes (unlike mammals), we can eliminate an entire subfamily with a mutation to a single gene. Here, we have examined the effect of eliminating each of three subfamilies. Such a task is presently impossible to accomplish in any mammalian system.

Animals↗
PubMed1995

A multigene family of novel K+ channels from Paramecium tetraurelia.

Paramecium tetraurelia has an excitable membrane with electrical properties resembling those of nerve and muscle cells from metazoans. However, the relationship between the molecules underlying electrical excitability in Paramecium and those found in metazoans has been unexplored. Using a PCR-based screen, we identified a multigene family of putative potassium channels in the ciliate protist Paramecium tetraurelia. Two distinct genomic clones were isolated, each encoding an apparently complete channel, Pak1 and Pak2. Fragments of three additional genes were also isolated. The deduced amino acid sequences appear to define a family of novel K+ channel genes which are only distantly related to metazoan potassium channels; the K+-selective pore region is exceptionally well conserved, but outside this region, PaK1 and PaK2 bear little resemblance to the metazoan voltage-gated K+ channels, Shaker Shal, Shab or Shaw. The uniqueness of this large set of K+ channel genes may be accounted for by the distinct evolutionary pressures at play in the single celled Paramecium versus the multicellular Metazoans. Since a large number of K+ channel mutants exist in Paramecium, the sequences of the K+ channel gene family reported here may represent a significant step towards the molecular characterization of these important behavioral mutants.

Amino Acid Sequence↗
PubMed1995

Multiple Shaker potassium channels in a primitive metazoan.

Voltage-gated potassium channels are critical elements in providing functional diversity in nervous systems. The diversity of voltage-gated K+ channels in modern triploblastic metazoans (such as mollusks, arthropods and vertebrates) is provided primarily by four gene subfamilies (Shaker, Shal, Shab, and Shaw), but there has been no data from the ancient diploblastic metazoans until now. Diploblasts, represented by jellyfish and other coelenterates, arose during the first major metazoan radiation and are the most structurally primitive animals to have true nervous systems. By comparing the K+ channels of diploblasts and triploblasts, we may determine the fundamental set of K+ channels present in the first nervous systems. We now report the isolation of two Shaker subfamily cDNA clones, jShak1 and jShak2, from the hydrozoan jellyfish Polyorchis penicillatus (Phylum Cnidaria). JShak1 and jShak2 express transient outward currents in Xenopus oocytes most similar to Shaker currents from Drosophila in their rates of inactivation and recovery from inactivation. The finding of multiple Shaker subfamily genes is significant in that multiple Shaker genes also exist in mammals. In Drosophila, multiple Shaker channels are also produced, but by a mechanism of alternative splicing. Thus, the Shaker K+ channel subfamily had an established functional identity prior to the first major radiation of metazoans, and multiple forms of Shaker channels have been independently selected for in a wide range of metazoans.

Amino Acid Sequence↗
PubMed1994

Calcium sensitivity of BK-type KCa channels determined by a separable domain.

High conductance, Ca(2+)-activated (BK-type) K+ channels from mouse (mSlo) and Drosophila (dSlo) differ in their functional properties but share a conserved core resembling voltage-gated K+ channels and a tail appended to the core by a nonconserved linker. We have found that the channel subunit is physically divisible into these two conserved domains and that the core determines such properties as channel open time, conductance, and, probably, voltage dependence, whereas the tail determines apparent Ca2+ sensitivity. Both domains are required for function. We demonstrated the different roles of the core and tail by taking advantage of the functional differences between mSlo and dSlo. Heterologous pairing of cores and tails from mSlo and dSlo showed that single-channel properties were always characteristic of the core species, but that apparent Ca2+ sensitivity was adjusted up or down depending on the species of the tail. Thus, the tail is implicated in the Ca(2+)-sensing role of BK channels.

Amino Acid Sequence↗
PubMed1994

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate.

The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.

Amino Acid Sequence↗
PubMed1994

Molecular evolution of K+ channels in primitive eukaryotes.

Cnidarians and ciliate protozoans represent evolutionary interesting phylogenetic groups for the study of K+ channel evolution. Cnidaria is a primitive metazoan phylum consisting of simple diploblast organisms which have few tissue types such as jellyfish, hydra, sea anemones, and corals. Their divergence from the rest of the metazoan line may predate the radiation of the major triploblast phyla by several hundred million years (Morris, 1993). Cnidarians are the most primitive metazoans to have an organized nervous system. Thus, comparing K+ channels cloned from cnidarians to those cloned from more advanced metazoans may reveal which types of K+ channel are most fundamental to electrical excitability in the nervous system. In contrast, channels in ciliate protozoans such as Paramecium may not have been designed to send electrical signals between cells, but simply to control the behavior, such as an avoidance reaction, of a single cell. Hence, comparing cloned Paramecium K+ channels to K+ channels cloned from cnidarians and other metazoans may reveal which types of K+ channel are most fundamental to electrical excitability in eukaryotes, and which K+ channels are specialized for neuronal signaling. Potassium channels are involved in a diversity of tasks and are universally present in eukaryotes. K+ channels set the resting membrane potentials of most metazoan and protozoan cells and are fundamental components of membrane electrical activity in virtually all eukaryotic systems. These channels control the shape, duration and frequency of metazoan action potentials and are known to participate in the action potentials of protozoans, fungi and plants as well (Hille, 1992). Voltage-clamp recordings have shown that a various assortment of voltage-gated K+ channels as well as Ca(2+)-activated K+ channels are widespread in eukaryotes (Hille, 1992). Thus, K+ channels appear to be crucial to behavioral responses in all classes of eukaryotes, including locomotion in metazoans and protozoans, and rapid growth responses and cell shape changes in plants. K+ channel diversity is by far the greatest in metazoans, which have made a strong commitment to electrically excitable cellular networks. There is an apparent need for a great diversity of K+ channel subtypes in these metazoans. Over 50 K+ channel sequences from many distinct gene families have been reported so far, and all but two (both from plants) have been found in triploblast metazoans. The complex needs of neuronal integration and neuromuscular transmission in triploblasts require exquisite control of cellular excitability. This is in large part achieved by an extensive and diverse set of K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

mSlo, a complex mouse gene encoding "maxi" calcium-activated potassium channels.

Complementary DNAs (cDNAs) from mSlo, a gene encoding calcium-activated potassium channels, were isolated from mouse brain and skeletal muscle, sequenced, and expressed in Xenopus oocytes. The mSlo-encoded channel resembled "maxi" or BK (high conductance) channel types; single channel conductance was 272 picosiemens with symmetrical potassium concentrations. Whole cell and single channel currents were blocked by charybdotoxin, iberiotoxin, and tetraethylammonium ion. A large number of variant mSlo cDNAs were isolated, indicating that several diverse mammalian BK channel types are produced by a single gene.

Alternative Splicing↗
PubMed1992

An essential 'set' of K+ channels conserved in flies, mice and humans.

The molecular genetic approach to studying K+ channels has revealed that at least four subfamilies of voltage-gated K+ channels originally discovered in Drosophila are conserved in mice and humans. This conservation of the K+ channel subfamilies Shaker, Shal, Shab, and Shaw suggests that not only the broad outlines of membrane electrical properties but also many molecular details as well evolved in the parent species ancestral to both invertebrate and vertebrate life. Shaker, Shal, Shab, and Shaw K+ channels have similar structures, but appear to be independent channel systems: when co-expressed in Xenopus oocytes, all four function independently. These four K+ channel subfamilies may be part of an essential 'set' of excitable channels required by most nervous systems. The task now remaining is to understand the functions of each member of the set.

Animals↗
PubMed1992

Genomic organization, nucleotide sequence, and cellular distribution of a Shaw-related potassium channel gene, Kv3.3, and mapping of Kv3.3 and Kv3.4 to human chromosomes 19 and 1.

Genomic and cDNA clones encoding a novel Shaw-related potassium channel gene have been isolated from mice and humans. The mouse-Kv3.3 gene encodes a protein of 679 amino acids. Unlike the vertebrate Shaker-related genes that have intronless coding regions, mouse Kv3.3 is encoded by at least two exons separated by 3 kb of intervening sequence. The amino-terminal 212 amino acids are encoded by a single exon, and the hydrophobic core of the protein beginning at the S1 transmembrane segment is contained in a separate exon. Multiple Kv3.3-hybridizing transcripts are visible in the mouse brain, liver, thymus, and heart. Using probes derived from a human genomic clone containing the 3' exon of human Kv3.3 (KCNC3), we have localized the gene to human chromosome 19. The related gene, human Kv3.4 (KCNC4), was localized to human chromosome 1.

Amino Acid Sequence↗
PubMed1991

mShal, a subfamily of A-type K+ channel cloned from mammalian brain.

We have cloned and expressed a mouse brain cDNA, mShal, that encodes a transient, A-type K+ current. mShal, the vertebrate homolog of the Drosophila Shal gene, defines a distinct subfamily of voltage-gated K+ channels. The Shal deduced proteins are more highly conserved between mouse and Drosophila than other presently known K+ channels. mShal carries a "low-threshold" A-type current with a hyperpolarized steady-state inactivation midpoint. Marked similarity was observed between mShal and its Drosophila homolog, fShal, with regard to voltage sensitivity of activation, macroscopic inactivation, steady-state inactivation, and 4-aminopyridine sensitivity. Sequence conservation for Shal proteins is unusually high at the amino terminus, an area considered important for inactivation. Removal of conserved amino-terminal residues from mShal modifies macroscopic inactivation but the transient nature of the current is preserved. Underlying the very high conservation of mShal and fShal may be a role in the nervous system that is conserved in widely divergent species.

Amino Acid Sequence↗
PubMed1991

Shaker, Shal, Shab, and Shaw express independent K+ current systems.

Although many K+ channel genes encoding homologous subunits have been cloned, a central question remains: how do these subunits associate to produce the diversity of K+ currents observed in living cells? Previous work has shown that different subunits encoded by the Shaker gene subfamily are able to form heteromultimers, which add to the diversity of currents. However, the unrestrained mixing of subunits from all genes to form hybrid channels would be undesirable for some cells that clearly require functionally discrete K+ currents. We show that Drosophila Shaker, Shal, Shab, and Shaw subunits form functional homomultimers, but that a molecular barrier to heteropolymerization is present. Coexpression of all four K+ channel systems does not alter their individual properties in any way. These experiments also demonstrate that multiple, independent A-current systems together with multiple, independent delayed rectifier systems can coexist in single cells.

Animals↗
PubMed1991

A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties.

We have cloned and expressed a mouse brain K+ channel that is the homolog of the Drosophila Shab K+ channel. Mouse and Drosophila Shab K+ channels (mShab and fShab, respectively) represent an instance of K+ channels and structurally related species that are both functionally and structurally conserved; most kinetic, voltage-sensitive, and pharmacological properties are similar for the 2 channels. The greatest functional difference between the currents is recovery from inactivation, which is several times slower for mShab than for fShab currents. In addition to conserved structure, the mShab polypeptide has an unusually long nonconserved region at the carboxyl end of the protein. Truncation of 293 residues from the carboxyl end produced no noticeable change in voltage-sensitive, kinetic, or pharmacological properties. Thus, the measured functional properties of mShab are determined by the remaining 564 residues, most of which are conserved. The mShab and fShab channels are naturally occurring structural variants having substitutions in conserved portions that appear relatively neutral with respect to all measured properties except for, possibly, the rate of recovery from inactivation. The mShab current closely resembles a native delayed-rectifier-type potassium current, IK, in hippocampal neurons.

Amino Acid Sequence↗
PubMed1990

K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse.

The Drosophila Shaker gene on the X chromosome has three sister genes, Shal, Shab, and Shaw, which map to the second and third chromosomes. This extended gene family encodes voltage-gated potassium channels with widely varying kinetics (rate of macroscopic current activation and inactivation) and voltage sensitivity of steady-state inactivation. The differences in the currents of the various gene products are greater than the differences produced by alternative splicing of the Shaker gene. In Drosophila, the transient (A current) subtype of the potassium channel (Shaker and Shal) and the delayed-rectifier subtype (Shab and Shaw) are encoded by homologous genes, and there is more than one gene for each subtype of channel. Homologs of Shaker, Shal, Shab, and Shaw are present in mammals; each Drosophila potassium-channel gene may be represented as a multigene subfamily in mammals.

Amino Acid Sequence↗