Voltage-sensitive sodium channels are responsible for the initiation and propagation of the action potential and therefore are important for neuronal excitability. Complementary DNA clones encoding the beta 1 subunit of the rat brain sodium channel were isolated by a combination of polymerase chain reaction and library screening techniques. The deduced primary structure indicates that the beta 1 subunit is a 22,851-dalton protein that contains a single putative transmembrane domain and four potential extracellular N-linked glycosylation sites, consistent with biochemical data. Northern blot analysis reveals a 1,400-nucleotide messenger RNA in rat brain, heart, skeletal muscle, and spinal cord. Coexpression of beta 1 subunits with alpha subunits increases the size of the peak sodium current, accelerates its inactivation, and shifts the voltage dependence of inactivation to more negative membrane potentials. These results indicate that the beta 1 subunit is crucial in the assembly, expression, and functional modulation of the heterotrimeric complex of the rat brain sodium channel.

Voltage-gated sodium channels in brain neurons are complexes of a pore-forming alpha subunit with smaller beta 1 and beta 2 subunits. cDNA cloning and sequencing showed that the beta 2 subunit is a 186 residue glycoprotein with an extracellular NH2-terminal domain containing an immunoglobulin-like fold with similarity to the neural cell adhesion molecule (CAM) contactin, a single transmembrane segment, and a small intracellular domain. Coexpression of beta 2 with alpha subunits in Xenopus oocytes increases functional expression, modulates gating, and causes up to a 4-fold increase in the capacitance of the oocyte, which results from an increase in the surface area of the plasma membrane microvilli. beta 2 subunits are unique among the auxiliary subunits of ion channels in combining channel modulation with a CAM motif and the ability to expand the cell membrane surface area. They may be important regulators of sodium channel expression and localization in neurons.

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: a (167 kDa), p (54 kDa), and y (30 kDa). Cleavage of disulfide bonds reveals two distinct a polypeptides and an additional component, 6.The a, subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). a2, a 143-kDa glycoprotein, has none of the properties characteristic of al but binds lectins and contains about 25% N-linked carbohydrate. a2 is disulfidelinked to 6, a 24-to 27-kDa glycopeptide. .3 (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. y (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purifiled anti-a, antibodies or a2-specific lentil lectin-agarose demonstrated that aja213yS behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the a26 complex dissociates from al.fy in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.

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We have studied the pathways by which extracellular bradykinin and adenosine 5'-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca2+]i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca2+]i as assessed by fura-2 based microfluorimetry. The bradykinin-induced response consisted of an initial Ca2+ mobilization from an internal pool followed by a sustained increase in [Ca2+]i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca2+ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca2+]i, due to an increase in the driving force for Ca2+ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca2+]i. Inward current and [Ca2+]i were enhanced during hyperpolarizing steps indicating a substantial Ca2+ influx through ATP-activated channels. No release of Ca2+ from internal stores, but a large Na+ inward current, was observed in Ca(2+)-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.