Shaker Protein Research Articles

Optically Mapping the Movement of Discrete Gating Charges in Shaker

The nature of the movement of the voltage sensor in voltage-gated ion channels has been a subject of intense study. In the canonical voltage-gated potassium channel Shaker, one major branch of experimentation has been to examine discrete gating charges in the voltage sensor, such as R362 (R1) or R365 (R2), to determine, via site-directed mutagenesis or cross-linking, the residues that they interact with, such as F290 or F416. However, site-directed mutagenesis may uncover indirect interactions rather than direct ones, while cross-linking may uncover rare states. We have aimed to track the movement of the charged residues directly, using a positively charged bimane derivative (qBBr) conjugated to a cysteine replacing either R362 or R365. Although bulkier than arginine, the distance of the positive charge from the protein backbone is similar, and the introduction of the dye is sufficient to restore typical gating current properties to these cysteine mutant constructs, strongly suggesting that qBBr is mimicking the natural function of the endogenous arginine. We have taken advantage of the properties of the bimane class of fluorescent dyes which are highly quenched by proximity to tyrosine and tryptophan but not other amino acids. Using qBBr attached to a chosen gating charge of Shaker proteins expressed in Xenopus laevis oocytes under voltage-clamp, we can optically measure the motion of a specific gating charge in relationship to a specific quenching site in response to changes in membrane potential. The quenching site can be either naturally occurring or artificially substituted into the protein. This method for optically mapping the movement of discrete gating charges in Shaker should be transferable to a wide variety of voltage-gated membrane proteins. Support: NIH GM030376.

Coordinating Different Homeostatic Processes

In this issue of Neuron, Bergquist and colleagues study a rapid form of presynaptic homeostatic regulation at the Drosophila neuromuscular junction. They show that the K(+) channel genes shal and shaker are reciprocally regulated in the central nervous system and suggest a hierarchical organization of intrinsic and synaptic homeostatic regulatory processes.

Identification of SLEEPLESS, a sleep-promoting factor.

Sleep is an essential process conserved from flies to humans. The importance of sleep is underscored by its tight homeostatic control. Through a forward genetic screen, we identified a gene, sleepless, required for sleep in Drosophila. The sleepless gene encodes a brain-enriched, glycosylphosphatidylinositol-anchored protein. Loss of SLEEPLESS protein caused an extreme (>80%) reduction in sleep; a moderate reduction in SLEEPLESS had minimal effects on baseline sleep but markedly reduced the amount of recovery sleep after sleep deprivation. Genetic and molecular analyses revealed that quiver, a mutation that impairs Shaker-dependent potassium current, is an allele of sleepless. Consistent with this finding, Shaker protein levels were reduced in sleepless mutants. We propose that SLEEPLESS is a signaling molecule that connects sleep drive to lowered membrane excitability.

Transient calnexin interaction confers long-term stability on folded K+ channel protein in the ER.

We recently showed that an unglycosylated form of the Shaker potassium channel protein is retained in the endoplasmic reticulum (ER) and degraded by proteasomes in mammalian cells despite apparently normal folding and assembly. These results suggest that channel proteins with a native structure can be substrates for ER-associated degradation. We have now tested this hypothesis using the wild-type Shaker protein. Wild-type Shaker is degraded by cytoplasmic proteasomes when it is trapped in the ER and prevented from interacting with calnexin. Neither condition alone is sufficient to destabilize the protein. Proteasomal degradation of the wild-type protein is abolished when ER mannosidase I trimming of the core glycan is inhibited. Our results indicate that transient interaction with calnexin provides long-term protection from ER-associated degradation.

Glycosylation Increases Potassium Channel Stability and Surface Expression in Mammalian Cells

N-linked glycosylation is not required for the cell surface expression of functional Shaker potassium channels in Xenopus oocytes (Santacruz-Toloza, L., Huang, Y., John, S. A., and Papazian, D. M. (1994) Biochemistry 33, 5607-5613). We have now investigated whether glycosylation increases the stability, cell surface expression, and proper folding of Shaker protein expressed in mammalian cells. The turnover rates of wild-type protein and an unglycosylated mutant (N259Q,N263Q) were compared in pulse-chase experiments. The wild-type protein was stable, showing little degradation after 48 h. In contrast, the unglycosylated mutant was rapidly degraded (t(1/2) = approximately 18 h). Lactacystin slowed the degradation of the mutant protein, implicating cytoplasmic proteasomes in its turnover. Rapid lactacystin-sensitive degradation could be conferred on wild-type Shaker by a glycosylation inhibitor. Expression of the unglycosylated mutant on the cell surface, assessed using immunofluorescence microscopy and biotinylation, was dramatically reduced compared with wild type. Folding and assembly were analyzed by oxidizing intersubunit disulfide bonds, which provides a fortuitous hallmark of the native structure. Surprisingly, formation of disulfide-bonded adducts was quantitatively similar in the wild-type and unglycosylated mutant proteins. Our results indicate that glycosylation increases the stability and cell surface expression of Shaker protein but has little effect on acquisition of the native structure.

Close Association of the N Terminus of Kv1.3 with the Pore Region

The Shaker superfamily encodes voltage-gated potassium (Kv) channels. The N termini of Shaker proteins are located intracellularly and contain several domains shown to regulate important aspects of channel function, such as speed of inactivation, channel assembly (T1 domain), and steady state protein level (T0 domain, amino acids 3-39 in rabbit). Mutations and/or deletion of certain amino acids in the T0 domain lead to a 13-fold amplification of Kv current as compared with wild type channels, primarily by increasing the absolute number of channel proteins present in the membrane (Segal, A. S., Yao, X., and Desir, G. V. (1999) Biochem. Biophys. Res. Commun. 254, 54-64). Although T0 mutants have kinetic properties virtually indistinguishable from wild type, they were noted to have a slightly larger single channel conductance, suggesting that the T0 domain might also interact with the pore region. In the present study we show that although T0 does not affect pore selectivity, it does modulate the binding affinity of the pore blocker, charybdotoxin. These results suggest that the N terminus of Kv1.3 is closely associated with the pore region.

Enhanced neurotransmitter release is associated with reduction of neuronal branching in a Drosophila mutant overexpressing frequenin.

Frequenin is a Drosophila Ca2+ binding protein whose overexpression causes a chronic facilitation of transmitter release at the larval neuromuscular junction and multiple firing of action potentials. These functional abnormalities are similar to those found in other hyperexcitable mutants (Shaker, ether-a-gogo, Hyperkinetic) which, in turn, exhibit increased branching at the motor nerve endings. We report here that mutants which overexpress frequenin have motor nerve terminals with reduced number and length of branches as well as number of synaptic boutons. Similar defects are observed in transgenic flies which have additional copies of the frequenin gene indicating that the phenotype can be adscribed to the overexpression of the protein. The ultrastructure of boutons, however, appears indistinguishable from wild type. In addition, we show here that frequenin overexpression leads also to a down regulation of Shaker proteins expression. The contrast between the observations in frequenin and the other hyperexcitable mutants indicates that nerve terminal morphology and enhanced transmitter release do not have a direct causal relationship.

Potassium Channel ä and β Subunits Assemble in the Endoplasmic Reticulum

We have characterized the maturation of Shaker K+ channel protein and the cellular site of assembly of pore-forming alpha and cytoplasmic beta subunits in a transfected mammalian cell line. Shaker protein is made as a partially glycosylated, immature precursor that is converted to a fully glycosylated, mature product. Shaker protein did not mature when transport from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Consistent with this finding, only the immature form was sensitive to digestion with endoglycosidase H. These results indicate that the immature protein is core-glycosylated in the ER, whereas the oligosaccharides of the mature protein have been further processed in the Golgi compartment. After inhibiting ER-to-Golgi transport, the oligomeric state of Shaker subunits was assessed by cross-linking in intact cells or by solubilization and sucrose gradient sedimentation. The results indicate that Shaker subunits assemble with each other in the ER. When co-expressed, the Kvbeta2 subunit also associated with Shaker in the ER. Assembly with the beta2 subunit did not increase the rate or extent of Shaker protein maturation. Our results indicate that the biogenesis of Shaker K+ channels in vivo involves core glycosylation and subunit assembly in the ER, followed by efficient transfer to the Golgi apparatus where the oligosaccharides are modified.

Essential Role fordlgin Synaptic Clustering of Shaker K+ChannelsIn Vivo

The assemblage of specific ion channels and receptors at synaptic sites is crucial for signaling between pre- and postsynaptic cells. However, the mechanisms by which proteins are targeted to and clustered at synapses are poorly understood. Here we show that the product of the Drosophila discs-large gene, DLG, is colocalized with Shaker K+ channels, which are clustered at glutamatergic synapses at the larval neuromuscular junction. In heterologous cells, DLG can cluster Shaker-type K+ channels, and, in the yeast two-hybrid system, the DLG PDZ1-2 domains bind directly to the C-terminal tail of Shaker proteins. We also demonstrate that DLG-Shaker interactions are required in vivo for Shaker clustering at the neuromuscular junction. Synaptic clustering of Shaker channels is abolished not only by mutations in dlg but also by a mutation in Shaker that deletes its C-terminal DLG binding motif. Analyses of various dlg mutant alleles suggest that channel clustering and synaptic targeting functions depend on distinct DLG domains. These studies demonstrate for the first time that DLG plays an important role in synaptic organization in vivo that correlates with its ability to bind directly to specific membrane proteins of the synapse.

Intersubunit interaction between amino- and carboxyl-terminal cysteine residues in tetrameric shaker K+ channels.

Shaker potassium (K+) channels normally lack intrasubunit and intersubunit disulfide bonds. However, disulfide bonds are formed between Shaker subunits in intact cells exposed to oxidizing conditions. Upon electrophoresis under nonreducing conditions, intersubunit disulfide bond formation was detected by the presence of four high molecular weight adducts of Shaker protein. This result suggests that intracellular cysteine residues are in sufficiently close proximity in the native structure of the Shaker channel to form intersubunit disulfide bonds. To test this hypothesis, wild-type and mutant Shaker proteins were exposed to oxidizing conditions in intact cells. Intersubunit disulfide bond formation was eliminated upon serine substitution of either C96 in the amino terminal or C505 in the carboxyl terminal of the protein. In contrast, disulfide bond formation was not eliminated upon serine substitution of both C301 and C308 in the cytoplasmic loop between transmembrane segments S2 and S3. Exposure of Shaker-expressing cells to oxidizing conditions did not significantly alter the amplitude, kinetics, or voltage dependence of the Shaker current, demonstrating that the native tertiary and quaternary structures of the channel were maintained under oxidizing conditions. These results indicate that intersubunit disulfide bonds form between C96 and C505, providing evidence that the amino- and carboxyl-terminal regions of adjacent subunits are in proximity in the native structure of the channel. The disulfide-bonded adducts were found to represent a dimer, a trimer, and two forms of tetramer, one linear and one circular, containing one, two, three, or four disulfide bonds, respectively. These results provide a direct biochemical demonstration that Shaker K+ channels contain four pore-forming subunits.

A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA). It has a rectifying current-voltage relationship and displays subconductance states, the largest of which amounts to A approximately equal to 90 pS. The channel is blocked by Cs- ions and gating requires the presence of Mg2+ ions. The kcsA gene has been identified in the gram-positive soil bacterium Streptomyces lividans. It encodes a predicted 17.6 kDa protein with two potential membrane-spanning helices linked by a central domain which shares a high degree of similarity with the H5 segment conserved among eukaryotic ion channels. Multiple alignments of deduced amino acids suggest that the novel channel has the closest kinship to the S5, H5 and S6 regions of voltage-gated K+ channel families, mainly to the subfamily represented by the Shaker protein from Drosophila melanogaster. Moreover, KcsA is most distantly related to eukaryotic inwardly rectifying channels with two putative predicted transmembrane segments.

Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K + channel subunit proteins

We are analyzing features of the K + channel subunit proteins that are critical for function and regulation of these proteins. Our studies show biochemically that subunit proteins from the Shaker and Shaw subfamilies fail to assemble into a heteromultimer. The basis for this incompatibility is the sequences contained within the T1 assembly domain. For a subunit protein to heteromultimerize with a Shaker subunit protein, two regions within the T1 domain, A and B, must be of the Shaker subtype. Finally, we show that the incompatibility of a Shaw A region for assembly with a Shaker protein depends upon the composition of a 30 amino acid conserved sequence in the A region.

Immunological Identification of the Shaker-Related Kv1.3 Potassium Channel Protein in T and B Lymphocytes, and Detection of Related Proteins in Flies and Yeast

Shaker-related potassium (K+) channel proteins contain sequences which exhibit remarkable conservation across species. We have generated polyclonal anti-peptide antibodies (Abs) which cross-react with peptide epitopes of several Shaker-related channels (Kv1.1, 1.2 and 1.3), in addition to a Kv1.3-specific Ab. The Kv1.3 -specific Abs react with a protein expressed in human T-cells (Jurkat and PBLs), as well as in mouse T-cells (EL-4) and pre-B cells (230.37). The cross-reactive Abs detect the Shaker protein in Drosophila melanogaster, in addition to an immunological1y related protein in the yeast Saccharomyces cerevisiae. Abs which recognize these shared epitopes could serve for the identification and biochemical characterization of Shaker-related proteins in diverse organisms.

Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells

Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.

Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.

Voltage-activated K+ channels are a family of closely related membrane proteins that differ in their gating behavior, conductance, and pharmacology. A prominent and physiologically important difference among K+ channels is their rate of inactivation. Inactivation rates range from milliseconds to seconds, and K+ channels with different inactivation properties have very different effects on signal integration and repetitive firing properties of neurons. The cloned Shaker B (H4) potassium channel is an example of a K+ channel that inactivates in a few milliseconds. Recent experiments have shown that removal of an N-terminal region of the Shaker protein by site-directed deletion practically abolishes this fast inactivation, but the modified channel does still inactivate during a prolonged depolarization lasting many seconds. Here we report that this remnant inactivation must occur by a distinct mechanism from the rapid inactivation of the wild-type Shaker channel. Like the inactivation of another K+ channel [Grissmer, S. & Calahan, M. (1989) Biophys. J. 55, 203-206], this slow inactivation is retarded by the application of a channel blocker, tetraethylammonium, to the extracellular side of the channel. By contrast, the fast inactivation of the wild-type Shaker channel is sensitive only to intracellular application of tetraethylammonium. Intracellular tetraethylammonium slows down the fast inactivation process, as though it competes with the binding of the inactivation particle.

Alternative Shaker transcripts express either rapidly inactivating or noninactivating K+ channels.

Two members of the Shaker K+ channel family designated ShA2 and ShD2 were characterized in the Xenopus oocyte expression system. The predicted amino acid sequences of ShA2 and ShD2 differ only in the amino terminus, which is located intracellularly according to the present topological model of K+ channels. The differing amino termini have profound effects on the electrophysiological and pharmacological properties of the K+ channel. Most markedly, the nature of the amino terminus determines whether the K+ channel mediates rapidly inactivating or noninactivating K+ currents. It also affects the 4-aminopyridine, tetraethylammonium, and charybdotoxin sensitivities of the K+ channels. These results suggest that the amino terminus of Shaker proteins affects K+ channel structures on both sides of the membrane.

A Family of Putative Potassium Channel Genes in Drosophila

Mutant flies in which the gene coding for the Shaker potassium channel is deleted still have potassium currents similar to those coded by the Shaker gene. This suggests the presence of a family of Shaker-like genes in Drosophila. By using a Shaker complementary DNA probe and low-stringency hybridization, three additional family members have now been isolated, Shab, Shaw, and Shal. The Shaker family genes are not clustered in the genome. The deduced proteins of Shab, Shaw, and Shal have high homology to the Shaker protein; the sequence identity of the integral membrane portions is greater than 50 percent. These genes are organized similarly to Shaker in that only a single homology domain containing six presumed membrane-spanning segments common to all voltage-gated ion channels is coded by each messenger RNA. Thus, potassium channel diversity could result from an extended gene family, as well as from alternate splicing of the Shaker primary transcript.

Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems.

Voltage-sensitive potassium channels are found in vertebrate and invertebrate central nervous systems. We have isolated a rat brain cDNA by cross-hybridization with a probe of the Drosophila Shaker gene complex. Structural conservation of domains of the deduced protein indicate that the rat brain cDNA encodes a voltage-sensitive potassium channel. Of the deduced amino acid sequence, 82% is homologous to the Drosophila Shaker protein indicating that voltage-sensitive potassium channels have been highly conserved during evolution. Selective pressure was highest on sequences facing the intracellular side and on proposed transmembrane segments S4-S6, suggesting that these domains are crucial for voltage-dependent potassium channel function. The corresponding rat mRNA apparently belongs to a family of mRNA molecules which are preferentially expressed in the central nervous system.

Cloning of a probable potassium channel gene from mouse brain

Potassium channels comprise a diverse class of ion channels important for neuronal excitability and plasticity. The recent cloning of the Shaker locus from Drosophila melanogaster has provided a starting point for molecular studies of potassium channels. Predicted Shaker proteins appear to be integral membrane proteins and have a sequence similar to the sequence of the S4 segment of the vertebrate sodium channel, where the S4 segment has been proposed to be the voltage sensor. Expression studies in frog oocytes confirm that Shaker encodes a component of a potassium channel (the A channel) that conducts a fast transient potassium current. Here we report the isolation of complementary DNA clones from the mouse brain, the nucleotide sequences of which predict a protein remarkably similar to the Shaker protein. The strong conservation of the predicted protein sequence in flies and mammals suggests that these mouse clones encode a potassium channel component and that the conserved amino acids may be essential to some aspect of potassium channel function.

Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila.

The Shaker locus of Drosophila contains a very large transcription unit. It is expressed predominantly in the nervous system by multiple, differential as well as alternative, splicing mechanisms into different, but functionally related proteins. The structure of the Shaker transcription unit and the properties of the encoded Shaker protein family provide a molecular basis for A channel diversity in excitable cells.