William S. Agnew

Primary structure and functional expression of a mammalian skeletal muscle sodium channel

We describe the isolation and characterization of a cDNA encoding the alpha subunit of a new voltage-sensitive sodium channel, microI, from rat skeletal muscle. The 1840 amino acid microI peptide is homologous to alpha subunits from rat brain, but, like the protein from eel electroplax, lacks an extended (approximately 200) amino acid segment between homologous domains I and II. Northern blot analysis indicates that the 8.5 kb microI transcript is preferentially expressed in skeletal muscle. Sodium channels expressed in Xenopus oocytes from synthetic RNA encoding microI are blocked by tetrodotoxin and mu-conotoxin at concentrations near 5 nM. The expressed sodium channels have gating kinetics similar to the native channels in rat muscle fibers, except that inactivation occurs more slowly.

μl Na+ channels expressed transiently in human embryonic kidney cells: Biochemical and biophysical properties

We describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.

Multiple gating modes and the effect of modulating factors on the μI sodium channel

Macroscopic current from the microI skeletal muscle sodium channel expressed in Xenopus oocytes shows inactivation with two exponential components. The major, slower components amplitude decreases with rapid pulsing. When microI cRNA is coinjected with rat skeletal muscle or brain mRNA the faster component becomes predominant. Individual microI channels switch between two principal gating modes, opening either only once per depolarization, or repeatedly in long bursts. These two modes differ in both activation and inactivation kinetics. There is also evidence for additional gating modes. It appears that the equilibrium among gating modes is influenced by a modulating factor encoded in rat skeletal muscle and brain mRNA. The modal gating is similar to that observed in hyperkalemic periodic paralysis.

Multiple oligosaccharide chains in the voltage-sensitive Na channel from Electrophorus electricus: Evidence for α-2,8-linked polysialic acid

Carbohydrate substituents on the large peptide of the voltage-sensitive Na channel from Electrophorus electricus electroplax have been partially characterized by their sensitivity to endoglycosidases H and F, peptide:N-glycosidase F, Endo-N-acetylneuraminidase, and to neuraminidase. The results suggest the presence of at least two classes of oligosaccharides: neutral, high mannose or hybrid oligosaccharides, and acidic, complex oligosaccharides with a core-structure terminating in an unbranched homopolymer of sialosyl units in alpha-2,8 linkages (much greater than 5 tandem sialic acids). Large decreases in apparent Mr produced by sialidase treatments suggest an extended carbohydrate structure that could inhibit protein-protein interaction. Polysialic acid was formerly proposed to be a unique constituent of neural cell adhesion molecules (N-CAMs) in vertebrates. However, ratios of sialic acid to galactose reported for mammalian brain and muscle Na channels suggest they may also carry this oligosaccharide.

Regulation of muscle sodium channel transcripts during development and in response to denervation.

We have recently described the cloning and functional expression of a new sodium channel subtype, microI, isolated from a denervated rat skeletal muscle cDNA library. In studies described here, we have used RNase protection and Northern blot analyses to examine the expression of microI mRNA in different tissues and in neonatal, adult, and adult denervated muscle. We found that microI transcripts were not expressed in brain or heart, or in the myogenic cell line L6, even after differentiation to myotubes. Transcripts for microI were present at low levels in neonatal skeletal muscle and increased to maximum levels in adult tissue, paralleling the expression of tetrodotoxin (TTX)-sensitive sodium currents. Surprisingly, denervation of adult muscle was also followed by a rise in microI mRNA, at a time when TTX-insensitive currents reappear. These results show that expression of this channel subtype is regulated by tissue type, development, and innervation.

The μI skeletal muscle sodium channel: Mutation E403Q eliminates sensitivity to tetrodotoxin but not to μ-conotoxins GIIIA and GIIIB

Voltage-sensitive Na channels from nerve and muscle are blocked by the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX). Mutagenesis studies of brain RII channels have shown that glutamate 387 (E387) is essential for current block by these toxins. We demonstrate here that mutation of glutamate 403 (E403) of the adult skeletal muscle μI channel (corresponding to E387 of RII) also prevents current blockade by TTX and STX, and by neo-saxitoxin. However, the mutation fails to prevent blockade by the peptide neurotoxins, μ-conotoxin GIIIA and GIIIB; these toxins are thought to bind to the same or overlapping sites with TTX and STX. The E403Q mutation may have utility as a marker for exogenous Na channels in transgenic expression studies, since there are no known native channels with the same pharmacological profile.

Fusion of native or reconstituted membranes to liposomes, optimized for single channel recording

We here describe a protocol for fusing vesicles into large structures suitable for patch clamp recording. The method may be used with native membrane vesicles or with liposomes containing reconstituted/purified ion channels. The resulting unilamellar membranes exhibit high channel surface abundance, yielding multiple channels in the average excised patch. The procedure has been used to record voltage-sensitive Na channels from three native membrane preparations (eel electroplax, rat skeletal muscle, squid optic nerve), and from reconstituted protein purified from eel electroplax. Channels treated with batrachotoxin (BTX) displayed characteristic activation voltage dependence, conductances, selectivity, and sensitivity to saxitoxin (STX).

Voltage-sensitive Na+ channels: motifs, modes and modulation.

Much recent progress has been made in understanding the structural organization and functional properties of voltage-dependent Na+ channels, in particular in the areas of activation, ion conductance, and inactivation. At the same time, however, electrophysiological studies have revealed new, more complex functional properties in the form of at least two gating modes and the existence of as yet unidentified modulatory factors.

A rapid ion-exchange assay for detergent-solubilized inositol 1,4,5-trisphosphate receptors

We describe a rapid ion-exchange syringe assay for [3H]inositol 1,4,5-trisphosphate binding to detergent-solubilized receptors. In extracts of rat cerebellar membranes, the assay resolves rapidly dissociating ligand complexes, detecting two to three times higher receptor abundance than conventional gel filtration spun column assays, and provides evidence for two classes of IP3-binding sites, representing 0.5-1.0% of total cerebellar membrane protein. Receptors purified from bovine and rat cerebellum exhibit a single class of high-affinity sites, with equilibrium dissociation constants (Kd = 4-8 nM) reflecting 20 to 25-fold higher affinity than reported in studies with spun-column methods.

[41] Squalene synthetase

Publisher Summary This chapter describes methods pertinent to the assay of the reactions in squalene synthesis. The enzyme may be solubilized and depleted of membrane lipids, with loss of catalytic activities. When reconstituted with the appropriate lipids, normal catalytic behavior can be restored. Methods for solubilization, delipidation, and reconstitution are given. Squalene synthetases are associated with the subcellular membranes in mammalian liver and in yeast. Although the enzyme and its products might initially seem to be of only moderate interest, pursuit of the reaction mechanisms and the underlying enzymology in very fundamental ways has stimulated a remarkable chapter in biochemical research. This has included the systematic resolution of all of the stereochemical transformations in the reactions between mevalonate and squalene. The rate of squalene synthesis seems more closely correlated with the rate of the condensation reaction than the amount of presqualene pyrophosphate present. The substrate “delivered” from the active site of the first reaction may be preferred over presqualene pyrophosphate accumulated in the membranes. Although these interpretations remain speculative, they are instructive in considering conditions for assay of the reactions and for solubilization and reconstitution of the enzyme.