napts, a mutation affecting sodium channel activity in Drosophila, is an allele of mle, a regulator of X chromosome transcription.

napts is a recessive mutation that affects the level of sodium channel activity and, at high temperature, causes paralysis associated with a loss of action potentials. We show, by genetic complementation tests, germline transformation, and analysis of mutations, that napts is a gain-of-function mutation of mle, a gene required for X chromosome dosage compensation and male viability. Molecular analyses of nap and mle mutations indicate that mle+, nap+, and napts activities are encoded by the same open reading frame and suggest that napts is due to a single amino acid substitution. Although napts is known to act via para+, an X-linked sodium channel structural gene, its effect is not due to a simple defect in para+ dosage compensation.     

Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases.     

Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides

A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.     

Three routes to suppression of the neurodegenerative phenotypes caused by Kinesin heavy chain mutations

Kinesin-1 is a motor protein that moves stepwise along microtubules by employing dimerized kinesin heavy chain (Khc) subunits that alternate cycles of microtubule binding, conformational change, and ATP hydrolysis. Mutations in the Drosophila Khc gene are known to cause distal paralysis and lethality preceded by the occurrence of dystrophic axon terminals, reduced axonal transport, organelle-filled axonal swellings, and impaired action potential propagation. Mutations in the equivalent human gene, Kif5A, result in similar problems that cause hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth type 2 (CMT2) distal neuropathies. By comparing the phenotypes and the complementation behaviors of a large set of Khc missense alleles, including one that is identical to a human Kif5A HSP allele, we identified three routes to suppression of Khc phenotypes: nutrient restriction, genetic background manipulation, and a remarkable intramolecular complementation between mutations known or likely to cause reciprocal changes in the rate of microtubule-stimulated ADP release by kinesin-1. Our results reveal the value of large-scale complementation analysis for gaining insight into protein structure-function relationships in vivo and point to possible paths for suppressing symptoms of HSP and related distal neuropathies.     

Dosage Effects of a Drosophila Sodium Channel Gene on Behavior and Axonal Excitability

The effects of para mutations on behavior and axonal excitability in Drosophila suggested that para specifically affects sodium channels. This hypothesis was confirmed by molecular analysis of the para locus, which demonstrates that the encoded para product is a sodium channel polypeptide. Here we characterize the effects of altered para+ dosage on behavior and axonal excitability, both in an otherwise wild-type background and in combination with two other mutations: napts, which also affects sodium channels, and ShKS133, which specifically affects potassium channels. Whereas it was previously shown that decreased dosage of para+ is unconditionally lethal in a napts background, we find that increased dosage of para+ suppresses napts. Similarly, we find that para hypomorphs or decreased dosage of para+ suppresses ShKS133, whereas increased dosage of para+ enhances ShKS133). The electrophysiological basis for these effects is investigated. Other genes in Drosophila that have sequence homology to sodium channels do not show such dosage effects, which suggests that the para+ product has a function distinct from that of other putative Drosophila sodium channel genes. We conclude that the number of sodium channels present in at least some Drosophila neurons can be affected by changes in para+ gene dosage, and that the level of para+ expression can strongly influence neuronal excitability.     

Effect of sodium channel abundance on Drosophila development, reproductive capacity and aging

The voltage-gated Na (+) channels (VGSC) are complex membrane proteins responsible for generation and propagation of the electrical signals through the brain, the skeletal muscle and the heart. The levels of sodium channels affect behavior and physical activity. This is illustrated by the maleless mutant allele (mle (napts)) in Drosophila, where the decreased levels of voltage-gated Na(+) channels cause temperature-sensitive paralysis. Here, we report that mle (napts) mutant flies exhibit developmental lethality, decreased fecundity and increased neurodegeneration. The negative effect of decreased levels of Na(+) channels on development and ts-paralysis was more pronounced at 18 and 29°C than at 25°C, suggesting particular sensitivity of the mle (napts) flies to temperatures above and below normal environmental conditions. Similarly, longevity of mle (napts) flies was unexpectedly short at 18 and 29°C compared with flies heterozygous for the mle (napts) mutation. Developmental lethality and neurodegeneration of mle (napts) flies was partially rescued by increasing the dosage of para, confirming a vital role of Na(+) channels in development, longevity and neurodegeneration of flies and their adaptation to temperatures.     

Biophysical defects in voltage.gated sodium channels associated with long QT and Brugada syndromes

The activity of voltage-gated sodium channels contributes to onset and duration of the cardiac action potential through an intricate balance with the activity of other ion channels. Activation of sodium channels leads to membrane depolarization and Phase 0 of the cardiac action potential. Sodium channel fast inactivation contributes to Phase 1, the initial repolarization. Slow inactivation and closed state fast inactivation determine channel availability and, thus, overall membrane excitability. Defects in any of these biophysical states or transitions between them, imparted by (over 170 reported thus far, including both Long QT3 and Brugada syndromes) mutations in the (over 2000) amino acids that compose the sodium channel protein, can lead to channel dysfunction that manifests as an abnormal cardiac action potential and electrocardiogram. A causal relationship between several such abnormalities and the panoply of sodium channel mutations have led to a greater understanding of the molecular underpinnings of cardiac arrhythmias as well as a deeper appreciation for the intricacies of sodium channel function. Here, we review the literature regarding these causal relationships from a perspective of the biophysical properties of sodium channels.     

TRPV4 links inflammatory signaling and neuroglial swelling

The activity of voltage-gated sodium channels contributes to onset and duration of the cardiac action potential through an intricate balance with the activity of other ion channels. Activation of sodium channels leads to membrane depolarization and Phase 0 of the cardiac action potential. Sodium channel fast inactivation contributes to Phase 1, the initial repolarization. Slow inactivation and closed state fast inactivation determine channel availability and, thus, overall membrane excitability. Defects in any of these biophysical states or transitions between them, imparted by (over 170 reported thus far, including both Long QT3 and Brugada syndromes) mutations in the (over 2000) amino acids that compose the sodium channel protein, can lead to channel dysfunction that manifests as an abnormal cardiac action potential and electrocardiogram. A causal relationship between several such abnormalities and the panoply of sodium channel mutations have led to a greater understanding of the molecular underpinnings of cardiac arrhythmias as well as a deeper appreciation for the intricacies of sodium channel function. Here, we review the literature regarding these causal relationships from a perspective of the biophysical properties of sodium channels.     

The pore helix dipole has a minor role in inward rectifier channel function

Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.     

Point mutations in domain III of a Drosophila neuronal Na channel confer resistance to allethrin

Voltage-gated sodium channels are the presumed site of action of pyrethroid insecticides and DDT. We screened several mutant sodium channel Drosophila lines for resistance to type I pyrethroids. In insecticidal bioassays the para(74) and para(DN7) fly lines showed greater than 4-fold resistance to allethrin relative to the allethrin sensitive Canton-S control line. The amino acid substitutions of both mutants are in domain III. The point mutation associated with para(74) lies within the S6 transmembrane region and the amino acid substitution associated with para(DN7) lies within the S4-S5 linker region. These sites are analogous to the mutations in domain II underlying knockdown resistance (kdr) and super-kdr, naturally occurring forms of pyrethroid resistance found in houseflies and other insects. Electrophysiological studies were performed on isolated Drosophila neurons from wild type and para(74) embryos placed in primary culture for three days to two weeks. The mutant para(74) sodium currents were kinetically similar to wild type currents, in activation, inactivation and time to peak. The only observed difference between para(74) and wild-type neurons was in the affinity of the type I pyrethroid, allethrin. Application of 500 nM allethrin caused removal of inactivation and prolonged tail currents in wild type sodium channels but had little or no effect on para(74) mutant sodium channels.     

Effect of channel block on the spiking activity of excitable membranes in a stochastic Hodgkin-Huxley model

The influence of intrinsic channel noise on the spontaneous spiking activity of poisoned excitable membrane patches is studied by use of a stochastic generalization of the Hodgkin-Huxley model. Internal noise stemming from the stochastic dynamics of individual ion channels is known to affect the collective properties of the whole ion channel cluster. For example, there exists an optimal size of the membrane patch for which the internal noise alone causes a regular spontaneous generation of action potentials. In addition to varying the size of ion channel clusters, living organisms may adapt the densities of ion channels in order to optimally regulate the spontaneous spiking activity. The influence of a channel block on the excitability of a membrane patch of a certain size is twofold: first, a variation of ion channel densities primarily yields a change of the conductance level; second, a down-regulation of working ion channels always increases the channel noise. While the former effect dominates in the case of sodium channel block resulting in a reduced spiking activity, the latter enhances the generation of spontaneous action potentials in the case of a tailored potassium channel blocking. Moreover, by blocking some portion of either potassium or sodium ion channels, it is possible to either increase or decrease the regularity of the spike train.     

Effects of channel blocking on information transmission and energy efficiency in squid giant axons

Action potentials are the information carriers of neural systems. The generation of action potentials involves the cooperative opening and closing of sodium and potassium channels. This process is metabolically expensive because the ions flowing through open channels need to be restored to maintain concentration gradients of these ions. Toxins like tetraethylammonium can block working ion channels, thus affecting the function and energy cost of neurons. In this paper, by computer simulation of the Hodgkin-Huxley neuron model, we studied the effects of channel blocking with toxins on the information transmission and energy efficiency in squid giant axons. We found that gradually blocking sodium channels will sequentially maximize the information transmission and energy efficiency of the axons, whereas moderate blocking of potassium channels will have little impact on the information transmission and will decrease the energy efficiency. Heavy blocking of potassium channels will cause self-sustained oscillation of membrane potentials. Simultaneously blocking sodium and potassium channels with the same ratio increases both information transmission and energy efficiency. Our results are in line with previous studies suggesting that information processing capacity and energy efficiency can be maximized by regulating the number of active ion channels, and this indicates a viable avenue for future experimentation.     

The microtubule plus-end tracking protein EB1 is required for Kv1 voltage-gated K+ channel axonal targeting

Axonal Kv1 channels regulate action potential propagation-an evolutionarily conserved function important for the control of motor behavior as evidenced from the linkage of human Kv1 channel mutations to myokymia/episodic ataxia type 1 (EA1) and the Shaker mutant phenotype in Drosophila. To search for the machinery that mediates axonal targeting of Kv1 channels composed of both alpha and beta subunits, we first demonstrate that Kvbeta2 is responsible for targeting Kv1 channels to the axon. Next, we show that Kvbeta2 axonal targeting depends on its ability to associate with the microtubule (MT) plus-end tracking protein (+TIP) EB1. Not only do Kvbeta2 and EB1 move in unison down the axon, Brefeldin A-sensitive Kv1-containing vesicles can also be found at microtubule ends near the cell membrane. In addition, we found that Kvbeta2 associates with KIF3/kinesin II as well. Indeed, Kv1 channels rely on both KIF3/kinesin II and EB1 for their axonal targeting.     

Abl tyrosine kinase and its substrate Ena/VASP have functional interactions with kinesin-1

Relatively little is known about how microtubule motors are controlled or about how the functions of different cytoskeletal systems are integrated. A yeast two-hybrid screen for proteins that bind to Drosophila Enabled (Ena), an actin polymerization factor that is negatively regulated by Abl tyrosine kinase, identified kinesin heavy chain (Khc), a member of the kinesin-1 subfamily of microtubule motors. Coimmunoprecipitation from Drosophila cytosol confirmed a physical interaction between Khc and Ena. Kinesin-1 motors can carry organelles and other macromolecular cargoes from neuronal cell bodies toward terminals in fast-axonal-transport. Ena distribution in larval axons was not affected by mutations in the Khc gene, suggesting that Ena is not itself a fast transport cargo of Drosophila kinesin-1. Genetic interaction tests showed that in a background sensitized by reduced Khc gene dosage, a reduction in Abl gene dosage caused distal paralysis and axonal swellings. A concomitant reduction in ena dosage rescued those defects. These results suggest that Ena/VASP, when not inhibited by the Abl pathway, can bind Khc and reduce its transport activity in axons.     

Channelopathies of inwardly rectifying potassium channels.

Mutations in genes encoding ion channels have increasingly been identified to cause disease conditions collectively termed channelopathies. Recognizing the molecular basis of an ion channel disease has provided new opportunities for screening, early diagnosis, and therapy of such conditions. This synopsis provides an overview of progress in the identification of molecular defects in inwardly rectifying potassium (Kir) channels. Structurally and functionally distinct from other channel families, Kir channels are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. In humans, persistent hyperinsulinemic hypoglycemia of infancy, a disorder affecting the function of pancreatic beta cells, and Bartter's syndrome, characterized by hypokalemic alkalosis, hypercalciuria, increased serum aldosterone, and plasma renin activity, are the two major diseases linked so far to mutations in a Kir channel or associated protein. In addition, the weaver phenotype, a neurological disorder in mice, has also been associated with mutations in a Kir channel subtype. Further genetic linkage analysis and full understanding of the consequence that a defect in a Kir channel would have on disease pathogenesis are among the priorities in this emerging field of molecular medicine.     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.