Interactions of key charged residues contributing to selective block of neuronal sodium channels by µ-conotoxin KIIIA.

Voltage-gated sodium channels are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias (Na(v)1.5), myotonias (Na(v)1.4), epilepsies (Na(v)1.2), and pain (Na(v)1.7). Thus, there is strong motivation to identify isoform-specific blockers and the molecular determinants underlying their selectivity among these channels. µ-Conotoxin KIIIA blocks rNa(v)1.2 (IC(50), 5 nM), rNa(v)1.4 (37 nM), and hNa(v)1.7 (97 nM), expressed in mammalian cells, with high affinity and a maximal block at saturating concentrations of 90 to 95%. Mutations of charged residues on both the toxin and channel modulate the maximal block and/or affinity of KIIIA. Two toxin substitutions, K7A and R10A, modulate the maximal block (52-70%). KIIIA-H12A and R14A were the only derivatives tested that altered Na(v) isoform specificity. KIIIA-R14A showed the highest affinity for Na(v)1.7, a channel involved in pain signaling. Wild-type KIIIA has a 2-fold higher affinity for Na(v)1.4 than for Na(v)1.7, which can be attributed to a missing outer vestibule charge in domain III of Na(v)1.7. Reciprocal mutations Na(v)1.4 D1241I and Na(v)1.7 I1410D remove the affinity differences between these two channels for wild-type KIIIA without affecting their affinities for KIIIA-R14A. KIIIA is the first µ-conotoxin to show enhanced activity as pH is lowered, apparently resulting from titration of the free N terminus. Removal of this free amino group reduced the pH sensitivity by 10-fold. Recognition of these molecular determinants of KIIIA block may facilitate further development of subtype-specific, sodium channel blockers to treat hyperexcitability disorders.

Structure-function relationships of the NCKX2 Na+/Ca2+-K+ exchanger.

K+-dependent Na+/Ca2+ exchangers (NCKX) have been shown to play important roles in physiological processes as diverse as phototransduction in rod photoreceptors, motor learning and memory in mice, and skin pigmentation in humans. Most structure-function studies on NCKX proteins have been carried out on the NCKX2 isoform, but sequence similarity suggests that the results obtained with the NCKX2 isoform are likely to apply to all NCKX1-5 members of the human SLC24 gene family. Here we review our recent work on the NCKX2 protein concerning the topological arrangement of transmembrane segments carrying out cation transport, and concerning residues important for transport function and cation binding.

Potassium-dependent sodium-calcium exchange through the eye of the fly.

In this review, we describe the characterization of a Drosophila sodium/calcium-potassium exchanger, Nckx30C. Sodium/calcium (-potassium) exchangers (NCX and NCKX) are required for the rapid removal of calcium in excitable cells. The deduced protein topology for NCKX30C is similar to that of mammalian NCKX, with 5 hydrophobic domains in the amino terminus separated from 6 at the carboxy-terminal end by a large intracellular loop. NCKX30C functions as a potassium-dependent sodium-calcium exchanger and is expressed in adult neurons and during ventral nerve cord development in the embryo. Nckx30C is expressed in a dorsal/ventral pattern in the eye-antennal disc, suggesting that large fluxes of calcium may be occurring during imaginal disc development in the larvae. NCKX30C may play a critical role in modulating calcium during development as well as in the removal of calcium and maintenance of calcium homeostasis in adults.

Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3.

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

Cardiac ion channel expression and contractile function in mice with deletion of thyroid hormone receptor alpha or beta.

Cardiac myocytes express the two thyroid hormone receptors (T(3)Rs), T(3)Ralpha and T(3)Rbeta. However, which isoform contributes to specific, T(3)-induced alterations of cardiac function remains unclear. Here, we used individual T(3)R isoform knockout (KO) mice to study the effects of T(3)Ralpha and T(3)Rbeta in the heart. Our findings indicate that potassium channel genes that code for K(+) channels involved in action potential repolarization, like KV 4.2 and minK, are T(3)Ralpha targets. Both are markedly regulated by thyroid status. The recently identified cyclic nucleotide-gated channels, HCN2 and HCN4, are targets of T(3)Ralpha and are unchanged in a euthyroid T(3)Rbeta KO. However, these transcripts respond markedly to altered T(3) signaling concomitant with bradycardia in T(3)Ralpha KO and hypothyroid animals, as well as tachycardia in hyperthyroid T(3)Rss KO mice. SERCA2a and myosins are T(3) regulated and were also targets of T(3)Ralpha, and the papillary muscles of alphaKO animals showed a slowed rate of force development. Because of the absence of significant cardiac effects in euthyroid T(3)Rss KO mice, we determined messenger RNA levels for both T(3)Ralpha and T(3)Rss in the heart. We found that T(3)Rss is present at a 1:3 ratio to T(3)Ralpha1. We conclude that the cardiac phenotype regulated by T(3) is predominantly mediated by T(3)Ralpha and that the lack of T(3)Ralpha cannot be compensated by T(3)Rss in the heart.

Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels.

Potassium channels that are inhibited by internal ATP (K(ATP) channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K(ATP) channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K(ATP) channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K(ATP) channel isoforms suggests that this pathway may have wide significance for the physiological regulation of K(ATP) channels in various tissues and organelles.

Minimal domain requirement for cation transport by the potassium-dependent Na/Ca-K exchanger. Comparison with an NCKX paralog from Caenorhabditis elegans.

The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent (45)Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassium-dependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.

Cloning and characterization of a potassium-dependent sodium/calcium exchanger in Drosophila.

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.

Molecular identification of the ryanodine receptor pore-forming segment.

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca(2+) release measurements, [(3)H]ryanodine binding, and single channel recordings in planar lipid bilayers. We report here that single point mutations introduced into this region of the mouse cardiac ryanodine receptor reduce or abolish high affinity [(3)H]ryanodine binding. Single channel analysis revealed that a single substitution of alanine for glycine 4824 within this region reduced the single channel conductance by 97%, from 798 picosiemens (pS) for the wild type channel to 22 pS. The G4824A mutant channel was modulated by Ca(2+), Mg(2+), ATP, caffeine, ruthenium red, and ryanodine. Co-expression of the wild type and G4824A mutant proteins produced single channels that have intermediate unitary conductances of 516, 256, 176, and 60 pS. These data suggest that this conserved region constitutes an essential part of the ryanodine binding site and the channel conduction pathway of the ryanodine receptor.

cDNA cloning and functional expression of the dolphin retinal rod Na-Ca+K exchanger NCKX1: comparison with the functionally silent bovine NCKX1.

cDNAs of human and bovine retinal rod Na+-Ca2++K+ exchanger (NCKX1) have previously been cloned, but potassium-dependent Na-Ca exchange activity upon heterologous expression has not been demonstrated. We have cloned NCKX1 cDNA from dolphin, examined function upon transfection in HEK293 cells, and compared the dolphin sequence encoded by the cDNA with those of human and bovine. The dolphin NCKX1 cDNA encodes 1013 amino acid residues. Comparison to bovine and human NCKX1 revealed strong conservation in the transmembrane domains (>95%), but relatively low conservation in the large extracellular ( approximately 50%) and cytosolic (<50%) domains. The dolphin cytosolic domain differs from the bovine sequence by the absence of a stretch of 114 amino acids. HEK293 cells transfected with dolphin NCKX1 cDNA showed K+-dependent Na-Ca exchange in >95% of the experiments, whereas transfection with bovine NCKX1 yielded no function. The bovine NCKX1 phenotype was imparted on dolphin NCKX1 when the dolphin cytosolic loop was replaced by that from bovine. Conversely, deletion of 114 amino acids from the bovine sequence to match the dolphin sequence resulted in a mutant bovine NCKX1 which performed K+-dependent Na-Ca exchange. These results suggest that domains within the large cytosolic loop of NCKX1 control functional activity when expressed in heterologous systems.

Chromosomal localization and genomic organization of the human retinal rod Na-Ca+K exchanger.

The retinal rod Na-Ca+K exchanger is a unique calcium extrusion protein found only in the outer segments of retinal rod photoreceptors. Rod Na-Ca+K exchanger cDNA (NCKX1) has been cloned from bovine and human retinas. Here, we have used fluorescent in situ hybridization and radiation hybrid mapping to localize the human NCKX1 gene to chromosome 15q22. We have determined the genomic organization of human rod NCKX1 and found one intron in the 5' untranslated region and eight introns within the coding region.

cDNA cloning of the human retinal rod Na-Ca + K exchanger: comparison with a revised bovine sequence.

PURPOSE: To clone the complementary DNA of the human retinal rod Na-Ca + K exchanger. METHODS: A human retinal cDNA library was screened initially with a radiolabeled probe representing the entire bovine rod Na-Ca + K exchanger cDNA and subsequently with probes from polymerase chain reaction fragments of the human retinal rod Na-Ca + K exchanger obtained after the initial screen. Twelve positive clones were used to obtain the entire coding sequence of the human retinal rod Na-Ca + K exchanger. RESULTS: The cDNA of the human retinal rod Na-Ca + K exchanger codes for a protein of 1081 amino acids, which shows 64.3% overall identity with the bovine retinal rod Na-Ca + K exchanger at the amino acid level. The two sets of putative transmembrane-spanning domains and their short connecting loops showed the highest degree of identity (94%-95%), whereas the extracellular loop at the N terminus showed a 59% identity. The large cytosolic loop that bisects the two sets of transmembrane-spanning domains contained two large deletions in the human exchanger; the first deletion contains 18 amino acids, whereas the second deletion involves a series of repeats that are dominated by acidic amino acid residues observed in the bovine, but not in the human, sequence. The authors observed that the bovine sequence contains a ninth repeat in addition to the eight repeats of the published sequence. CONCLUSIONS: The authors cloned the cDNA of the human retinal rod Na-Ca + K exchanger as a first step in examining the possibility that this gene could be the locus of disease-causing mutations.

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5.

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).

ASE-1: a novel protein of the fibrillar centres of the nucleolus and nucleolus organizer region of mitotic chromosomes.

A novel nucleolar component has been identified and cloned using a human autoimmune serum. This antigen, as inferred from the cDNA sequence, is an Mr 55000 protein. Immuno blot analysis, however, of both the native protein and the in vitro translation products of the cDNA showed that they migrate on SDS-PAGE at an apparent molecular mass of 90000 A BLAST search using the cDNA sequence indicated that it is in an antisense orientation to and overlaps the gene of the DNA repair enzyme ERCC-1. An open reading frame, without a translational start site, had been observed by others in this region of the chromosome 19 (19q13.3) and the putative protein was termed ASE-1 (Anti-Sense to ERCC-1). Our cDNA is a full-length equivalent of that open reading frame. ASE-1 was found to contain two domains that are present in a number of nucleolar specific proteins originating from a variety of organisms: a glycine-, arginine- and phenylalanine-rich putative nucleotide interaction domain and an alternating basic/acidic region. Indirect immunofluorescence analysis using antibodies generated to cloned regions of ASE-1 indicated that this protein occurs at the fibrillar centres of the nucleolus in interphase, the putative sites of rDNA transcription, and during cell division it is localized to the nucleolus organizer regions of the chromosomes. ASE-1 co-localises with the RNA polymerase I transcription initiation factor UBF/NOR-90 throughout all stages of the cell cycle and these two proteins associate with each other in vitro.

The spindle kinesin-like protein HsEg5 is an autoantigen in systemic lupus erythematosus.

OBJECTIVE: Autoantibodies directed against the mitotic spindle apparatus (MSA) have been shown to target an antigen referred to as NuMA (nuclear mitotic apparatus). In this study, we identified a second MSA antigen as the spindle kinesin-like protein HsEg5. We studied the frequency of antibodies to HsEg5 in human sera that demonstrate the MSA pattern of staining, the frequency of autoantibodies to HsEg5 in patients with systemic lupus erythematosus (SLE), and the clinical features of patients with antibodies to HsEg5. METHODS: A prototype serum from an SLE patient was used to isolate a 4.8-kilobase complementary DNA (cDNA) from a HeLa cDNA library. Western blot, immunoprecipitation, and sequence analysis revealed that the antigen was an approximately 130-kd protein, HsEg5. The frequency of autoantibodies to recombinant HsEg5 in 51 sera that demonstrated an MSA pattern of staining on HEp-2 and HeLa cells was detected by immunoblotting 2 constructs of the cDNA. The clinical features of patients with antibodies directed against HsEg5 was obtained by retrospective chart review. RESULTS: The antigen responsible for the MSA-35 pattern was identified as the human kinesin-like protein HsEg5. Seven of 51 sera (14%) that demonstrated an MSA pattern of staining reacted with recombinant HsEg5. Six of 7 of the HsEg5-positive patients (86%) had SLE, and 1 had Sjögren's syndrome. The indirect immunofluorescent staining pattern of sera that reacted with HsEg5 could be distinguished from the other sera that reacted with NuMA. In an unselected cohort of 52 SLE patients, 3 (6%) had autoantibodies reactive with the recombinant HsEg5. CONCLUSION: Autoantibodies to MSA fall into 2 major classes: those reactive with NuMA and those reactive with HsEg5. Autoantibodies to HsEg5 are found in a lower frequency than NuMA in sera that demonstrate the MSA pattern of staining and appear to be specifically associated with SLE. HsEg5 can be distinguished from NuMA by indirect immunofluorescence and Western blotting.

The relationship of HsEg5 and the actin cytoskeleton to centrosome separation.

Although centrosome separation is essential to the formation of a bipolar spindle, it can proceed along several different pathways. This raises questions as to the similarity between the mechanism(s) underlying these various forms of separation. To address this question we reinvestigated centrosome separation in HeLa cells using a variety of techniques. We present a refined description of the two major pathways of centrosome separation found in HeLa cells and demonstrate that each of these pathways has its own timing, protein requirements, morphological characteristics, and relationship to spindle assembly. The first pathway, which occurs in prophase cells, is dependent on an intact actin cytoskeleton, and when this pathway is completed prior to nuclear envelope breakdown, the microtubules associated with this process do not become part of the spindle. Thus, centrosome separation and spindle pole organization can occur as two separate events. The second centrosome separation pathway is found in cells in which separation occurs concurrent with prometaphase. In this case, centrosome separation and the formation of the mitotic spindle are integrated together and an intact actin cytoskeleton is not required. The relationship between these multiple pathways of centrosome separation and the distribution of the human kinesin-like protein HsEg5 was also investigated. This protein was found associated with all centrosomal microtubules present during both prophase and prometaphase centrosome separation, as well as with prophase centrosomes displaying independent movement in Cytochalasin-D treated cells. In addition, we demonstrate that this protein is associated with post-mitotic centrosome movement which involves a single centrosome. Thus, HsEg5 is a feature of individual centrosome function and does not require anti-parallel microtubule arrays.

CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis.

Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events.

Evolution of protamine P1 genes in mammals.

Protamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that protamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5-6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Molecular phylogeny and evolution of marsupial protamine P1 genes.

We report the DNA sequences of protamine P1 genes and flanking regions from 21 mammalian species, including representatives of all extant marsupial orders. The protamine P1 locus in marsupials displays highly conserved 5' and 3' flanking sequences, as well as a highly variable intron. Marsupial protamines examined are distinct from those of eutherian mammals in lacking cysteine residues, a feature that may be correlated with lower stability of marsupial sperm nuclei during chromatin condensation. Phylogenetic analysis of protamine sequences leads to the following conclusions: (i) the microbiothere Dromiciops is part of a clade that includes dasyurids, Notoryctes and diprotodontians but not bandicoots; (ii) dasyurids are the closest living relatives of Notoryctes; (iii) macropodids, phalangerids and pseudocheirids form a clade apart from the phascolarctids; and (iv) the closest living relatives of caenolestids are didelphids.

A brain-specific activator of cyclin-dependent kinase 5.

Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref. 8). When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.