Vann Bennett

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier.

We have characterized a new ankyrin gene, expressed in brain and other tissues, that is subject to extensive tissue-specific alternative mRNA processing. The full-length polypeptide has a molecular mass of 480 kDa and includes a predicted globular head domain, with membrane- and spectrin-binding activities, as well as an extended “tail” domain. We term this gene ankyrinG based on its giant size and general expression. Two brain-specific isoforms of 480 kDa and 270 kDa were identified that contain a unique stretch of sequence highly enriched in serine and threonine residues immediately following the globular head domain. Antibodies against the serine-rich domain and spectrin-binding domain revealed labeling of nodes of Ranvier and axonal initial segments. Ankyrin-binding proteins also known to be localized in these specialized membrane domains include the voltage-dependent sodium channel, the sodium/potassium ATPase, sodium/calcium exchanger, and members of the neurofascin/L1 family of cell adhesion molecules. The neural-specific ankyrinG polypeptides are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to nodes of Ranvier and axonal initial segments.

A Common Ankyrin-G-Based Mechanism Retains KCNQ and NaV Channels at Electrically Active Domains of the Axon

KCNQ (KV7) potassium channels underlie subthreshold M-currents that stabilize the neuronal resting potential and prevent repetitive firing of action potentials. Here, antibodies against four different KCNQ2 and KCNQ3 polypeptide epitopes show these subunits concentrated at the axonal initial segment (AIS) and node of Ranvier. AIS concentration of KCNQ2 and KCNQ3, like that of voltage-gated sodium (NaV) channels, is abolished in ankyrin-G knock-out mice. A short motif, common to KCNQ2 and KCNQ3, mediates both in vivo ankyrin-G interaction and retention of the subunits at the AIS. This KCNQ2/KCNQ3 motif is nearly identical to the sequence on NaV α subunits that serves these functions. All identified NaV and KCNQ genes of worms, insects, and molluscs lack the ankyrin-G binding motif. In contrast, vertebrate orthologs of NaV α subunits, KCNQ2, and KCNQ3 (including from bony fish, birds, and mammals) all possess the motif. Thus, concerted ankyrin-G interaction with KCNQ and NaV channels appears to have arisen through convergent molecular evolution, after the division between invertebrate and vertebrate lineages, but before the appearance of the last common jawed vertebrate ancestor. This includes the historical period when myelin also evolved.

Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.

The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes

Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10–15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.

Nanospring behaviour of ankyrin repeats

Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel α-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17–29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.

Adducin: structure, function and regulation

Abstract. Adducin is a ubiquitously expressed membrane-skeletal protein localized at spectrin-actin junctions that binds calmodulin and is an in vivo substrate for protein kinase C (PKC) and Rho-associated kinase. Adducin is a tetramer comprised of either α/β or α/γ heterodimers. Adducin subunits are related in sequence and all contain an N-terminal globular head domain, a neck domain and a C-terminal protease-sensitive tail domain. The tail domains of all adducin subunits end with a highly conserved 22-residue myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that has homology to MARCKS protein. Adducin caps the fast-growing ends of actin filaments and also preferentially recruits spectrin to the ends of filaments. Both the neck and the MARCKS-related domains are required for these activities. The neck domain self-associates to form oligomers. The MARCKS-related domain binds calmodulin and contains the major phosphorylation site for PKC. Calmodulin, gelsolin and phosphorylation by the kinase inhibit in vitro activities of adducin involving actin and spectrin. Recent observations suggest a role for adducin in cell motility, and as a target for regulation by Rho-dependent and Ca2+-dependent pathways. Prominent physiological sites of regulation of adducin include dendritic spines of hippocampal neurons, platelets and growth cones of axons.

The spectrin-actin junction of erythrocyte membrane skeletons.

High-resolution electron microscopy of erythrocyte membrane skeletons has provided striking images of a regular lattice-like organization with five or six spectrin molecules attached to short actin filaments to form a sheet of five- and six-sided polygons. Visualization of the membrane skeletons has focused attention on the (spectrin)5,6-actin oligomers, which form the vertices of the polygons, as basic structural units of the lattice. Membrane skeletons and isolated junctional complexes contain four proteins that are stable components of this structure in the following ratios: 1 mol of spectrin dimer, 2-3 mol of actin, 1 mol of protein 4.1 and 0.1-0.5 mol of protein 4.9 (numbers refer to mobility on SDS gels). Additional proteins have been identified that are candidates to interact with the junction, based on in vitro assays, although they have not yet been localized to this structure and include: tropomyosin, tropomyosin-binding protein and adducin. The spectrin-actin complex with its associated proteins has a key structural role in mediating cross-linking of spectrin into the network of the membrane skeleton, and is a potential site for regulation of membrane properties. The purpose of this article is to review properties of known and potential constituent proteins of the spectrin-actin junction, regulation of their interactions, the role of junction proteins in erythrocyte membrane dysfunction, and to consider aspects of assembly of the junctions.

Regulation of the Association of Adducin with Actin Filaments by Rho-associated Kinase (Rho-kinase) and Myosin Phosphatase*

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted with adducinin vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated α-adducin in vitro and in vivo and that the phosphorylation of α-adducin by Rho-kinase enhanced the interaction of α-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward α-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.

Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain.

We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor “dyads.” E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B+/− cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.