Stephen C. Cannon

Expanded CUG Repeats Trigger Aberrant Splicing of ClC-1 Chloride Channel Pre-mRNA and Hyperexcitability of Skeletal Muscle in Myotonic Dystrophy

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.

A proposed neural network for the integrator of the oculomotor system

Single-unit recordings, stimulation studies, and eye movement measurements all indicate that the firing patterns of many oculomotor neurons in the brain stem encode eye-velocity commands in premotor circuits while the firing patterns of extraocular motoneurons contain both eye-velocity and eye-position components. It is necessary to propose that the eye-position component is generated from the eye-velocity signal by a leaky hold element or temporal integrator. Prior models of this integrator suffer from two important problems. Since cells appear to have a steady, background signal when eye position and velocity are zero, how does the integrator avoid integrating this background rate? Most models employ some form of lumped, oositive feedback the gain of which must be kept within totally unreasonable limits for proper operation. We propose a lateral inhibitory network of homogeneous neurons as a model for the neural integrator that solves both problems. Parameter sensitivity studies and lesion simulations are presented to demonstrate robustness of the model with respect to both the choice of parameter values and the consequences of pathological changes in a portion of the neural integrator pool.

Leukocyte Common Antigen-Related Phosphatase Is a Functional Receptor for Chondroitin Sulfate Proteoglycan Axon Growth Inhibitors

Chondroitin sulfate proteoglycans (CSPGs) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division, and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane leukocyte common antigen-related phosphatase (LAR). CSPGs bind LAR with high affinity in transfected COS-7 cells and coimmunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knock-out mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG–LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.

Functional expression of sodium channel mutations identified in families with periodic paralysis

Two mutations in the sodium channel alpha subunit that have been implicated as the cause of periodic paralysis were studied by functional expression in a mammalian cell line. Both mutations disrupted inactivation without affecting the time course of the onset of the sodium current or the single-channel conductance. This is the same functional defect that was observed in myotubes cultured from affected patients and proves that these mutations are not benign polymorphisms. Unlike the currents in the myotubes, however, there was no consistent potassium dependence for the noninactivating component. These mutations also define new regions of the sodium channel alpha subunit that are involved in the process of inactivation.

A sodium channel defect in hyperkalemic periodic paralysis: Potassium-induced failure of inactivation

Hyperkalemic periodic analysis (HPP) is an autosomal dominant disorder characterized by episodic weakness lasting minutes to days in association with a mild elevation in serum K+. In vitro measurements of whole-cell currents in HPP muscle have demonstrated a persistent, tetrodotoxin-sensitive Na+ current, and we have recently shown by linkage analysis that the Na+ channel alpha subunit gene may contain the HPP mutation. In this study, we have made patch-clamp recordings from cultured HPP myotubes and found a defect in the normal voltage-dependent inactivation of Na+ channels. Moderate elevation of extracellular K+ favors an aberrant gating mode in a small fraction of the channels that is characterized by persistent reopenings and prolonged dwell times in the open state. The Na+ current, through noninactivating channels, may cause the skeletal muscle weakness in HPP by depolarizing the cell, thereby inactivating normal Na+ channels, which are then unable to generate an action potential. Thus the dominant expression of HPP is manifest by inactivation of the wild-type Na+ channel through the influence of the mutant gene product on membrane voltage.

MOD-1 is a serotonin-gated chloride channel that modulates locomotory behaviour in C. elegans.

The neurotransmitter and neuromodulator serotonin (5-HT) functions by binding either to metabotropic G-protein-coupled receptors (for example, 5-HT 1, 5-HT2, 5-HT4 to 5-HT7), which mediate ‘slow’ modulatory responses through numerous second messenger pathways, or to the ionotropic 5-HT3 receptor, a non-selective cation channel that mediates ‘fast’ membrane depolarizations. Here we report that the gene mod-1 (for modulation of locomotion defective) from the nematode Caenorhabditis elegans encodes a new type of ionotropic 5-HT receptor, a 5-HT-gated chloride channel. The predicted MOD-1 protein is similar to members of the nicotinic acetylcholine receptor family of ligand-gated ion channels, in particular to GABA (γ-aminobutyric acid)- and glycine-gated chloride channels. The MOD-1 channel has distinctive ion selectivity and pharmacological properties. The reversal potential of the MOD-1 channel is dependent on the concentration of chloride ions but not of cations. The MOD-1 channel is not blocked by calcium ions or 5-HT 3a-specific antagonists but is inhibited by the metabotropic 5-HT receptor antagonists mianserin and methiothepin. mod-1 mutant animals are defective in a 5-HT-mediated experience-dependent behaviour and are resistant to exogenous 5-HT, confirming that MOD-1 functions as a 5-HT receptor in vivo.

A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle

Another micropeptide flexes its muscle Genome annotation is a complex but imperfect art. Attesting to its limitations is the growing evidence that certain transcripts annotated as long noncoding RNAs (lncRNAs) in fact code for small peptides with biologically important functions. One such lncRNA-derived micropeptide in mammals is myoregulin, which reduces muscle performance by inhibiting the activity of a key calcium pump. Nelson et al. describe the opposite activity in a second lncRNA-derived micropeptide in mammalian muscle, called DWORF (see the Perspective by Payre and Desplan). This peptide enhances muscle performance by activating the same calcium pump. DWORF may prove to be useful in improving the cardiac muscle function of mammals with heart disease. Science, this issue p. 271; see also p. 226 A long noncoding RNA encodes a small peptide that activates a calcium pump regulating muscle contraction. [Also see Perspective by Payre and Desplan] Muscle contraction depends on release of Ca2+ from the sarcoplasmic reticulum (SR) and reuptake by the Ca2+adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca2+ transient amplitude and SR Ca2+ load while reducing the time constant of cytosolic Ca2+ decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca2+ clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility.

Theoretical reconstruction of myotonia and paralysis caused by incomplete inactivation of sodium channels

Muscle fibers from individuals with hyperkalemic periodic paralysis generate repetitive trains of action potentials (myotonia) or large depolarizations and block of spike production (paralysis) when the extracellular K+ is elevated. These pathologic features are thought to arise from mutations of the sodium channel alpha subunit which cause a partial loss of inactivation (steady-state Popen approximately 0.02, compared to < 0.001 in normal channels). We present a model that provides a possible mechanism for how this small persistent sodium current leads to repetitive firing, why the integrity of the T-tubule system is required to produce myotonia, and why paralysis will occur when a slightly larger proportion of channels fails to inactivate. The model consists of a two-compartment system to simulate the surface and T-tubule membranes. When the steady-state sodium channel open probability exceeds 0.0075, trains of repetitive discharges occur in response to constant current injection. At the end of the current injection, the membrane potential may either return to the normal resting value, continue to discharge repetitive spikes, or settle to a new depolarized equilibrium potential. This after-response depends on both the proportion of noninactivating sodium channels and the magnitude of the activity-driven K+ accumulation in the T-tubular space. A reduced form of model is presented in which a two-dimensional phase-plane analysis shows graphically how this diversity of after-responses arises as extracellular [K+] and the proportion of noninactivating sodium channels are varied.

An improved neural-network model for the neural integrator of the oculomotor system: More realistic neuron behavior

The discharge rates of premotor, brain-stem neurons that create eye movements modulate in relation to eye velocity yet firing rates of extraocular motoneurons contain both eye-position and eyevelocity signals. The eye-position signal is derived from the eye-velocity command by means of a neural network which functioins as a temporal integrator. We have previously proposed a network of lateral-inhibitory neurons that is capable of performing the required integration. That analysis centered on the temporal aspects of the signal processing for a limited class of idealized inputs. All of its cells were identical and carried only the integrated signal. Recordings in the brain stem, however, show that neurons in the region of the neural integrator have a variety of background firing rates, all carry some eye-velocity signal as well as the eye-position signal, and carry the former with different strengths depending on the type of eye movement being made. It was necessary to see if the proposed model could be modified to make its neurons more realistic.By modifying the spatial distribution of afferents to the network, we demonstrate that the same basic model functions properly in spite of afferents with nonuniform background firing rates. To introduce the eye-velocity signal a double-layer network, consisting of inhibitory and excitatory cells, was necessary. By presenting the velocity input to only local regions of this network it was shown that all cells in the network still carried the integrated signal and that its cells could carry different eye-velocity signals for different types of eye movements. Thus, this model stimulates quantitatively and qualitatively, the behavior of neurons seen in the region of the neural integrator.

A Na+ Channel Mutation Linked to Hypokalemic Periodic Paralysis Exposes a Proton-selective Gating Pore

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca2+ channel) or NaV1.4 (the skeletal muscle voltage-gated Na+ channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice (“gating-pore”) in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K+ channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K+ and Cl− conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of VREST indirectly, for instance, by interfering with intracellular pH homeostasis.