Voltage-sensitive sodium channels: agents that perturb inactivation gating.

In summary, the voltage-sensitive sodium channel from eel electroplax provides an optimal preparation for biochemical and biophysical studies of molecular structure and gating. We have demonstrated that the purified and reconstituted protein is capable of functioning normally, exhibiting, among other properties, voltage-dependent activation and inactivation gating mechanisms. We have been able to recreate the classical electrophysiological studies in which inactivation gating can be removed by proteolytic modification of the cytoplasmic surface of the molecule, and have mapped the probable site of modification to the peptide segment lying between subunit domains III and IV.

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.

Reconstituted voltage-sensitive sodium channel from Electrophorus electricus: chemical modifications that alter regulation of ion permeability.

At equilibrium, voltage-sensitive sodium channels normally are closed at all potentials. They open transiently in response to changes in membrane voltage or chronically under the influence of certain neurotoxins. Covalent modifications that result in chronic opening may help identify molecular domains involved in conductance regulation.

Electrical stimulation of neurointermediate lobes of mice elicits calcium-dependent output of melanocyte-stimulating hormone.

Current pulses applied to isolated neurointermediate lobes of mice increased output of melanocyte-stimulating hormone. This response was dependent on extracellular calcium over a wide range of stimulus intensities and thus appears to be a true secretory response from the melanotrophs. Since substantial responses persisted in the presence of tetrodotoxin, much of the effect seems to be independent of Na spiking.

Fluorescence assay for neurotoxin-modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ.

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin.