L-type Ca2+ channels and purinergic P2X2 cation channels participate in calcium-tyrosine kinase-mediated PC12 growth cone arrest.

During development and regeneration of the nervous system, growth cones of the various nerve cells navigate and direct neurite elongation by detecting and responding to cues in the environment. To investigate changes in growth cone behaviour due to calcium influx we used nerve growth factor (NGF)-induced growth cones of PC12 (rat pheochromocytoma cells) cells as a model. High external concentrations of potassium and ATP depress growth cone motility, induce club-shaped growth cones and reduce filopodia length and the number and relative F-actin contents of single growth cones (r.a.c.), respectively. The cellular responses are mediated by a sustained increase in the intracellular free Ca2+ concentrations ([Ca2+]i) as monitored by calcium-sensitive fluorescent dyes and confocal microfluorimetry. The responses are not detectable in the presence of the protein tyrosine kinase inhibitor genistein. Immunocytochemistry revealed an increased level of tyrosine-phosphorylated proteins in cell bodies and growth cones but not in cell nuclei. Paxillin, a cytoskeleton-associated protein located in neurites and growth cones, was detected among the phosphotyrosine proteins. The sustained (> 30 s) Ca2+ influx through voltage-gated L-type but not N- or P-type Ca2+ channels induced the F-actin loss and tyrosine phosphorylation. Ca2+ entry through P2X2 ligand-gated channels caused the same effects. Our data suggest the following mechanism: increased [Ca2+]i levels activate tyrosine kinases located close to the ion channels which then leads to changes in morphology due to tyrosine phosphorylation of proteins, e. g. paxillin.

Annexin I modulates cell functions by controlling intracellular calcium release.

Annexin I is an intracellular protein in search of a function. Ex vivo it has calcium- and phospholipid-binding properties. To evaluate its role in vivo, MCF-7 cells were stably transfected with annexin I in sense or antisense orientations. In cells overexpressing annexin I, calcium release was abrogated on stimulation of purinergic or bradykinin receptors, whereas non-transfected cells or cells with down-regulated annexin I released calcium within seconds. Basal calcium and calcium stores were not affected. The impaired calcium release was paralleled by a down-regulation of the activities of phospholipase C, group II phospholipase A2, and E-cadherin with altered adhesion and enhanced tumor growth on soft agar. Significantly smaller tumors, with the histologically most differentiated cells, were observed in nude mice inoculated with cells transfected with the antisense rather than with the sense plasmid. These observations indicate that annexin I modulates cell functions by controlling intracellular calcium release. Frey, B. M., Reber, B. F. X., Vishwanath, B. S., Escher, G., Frey, F. J. Annexin I modulates cell functions by controlling intracellular calcium release.

Differential cytoskeletal changes during growth cone collapse in response to hSema III and thrombin.

Growth cones are known as the site of action of many factors that influence neurite growth behavior. To assess how different collapsing agents influence the growth cone cytoskeleton, we used recombinant human Semaphorin III (hSema III) and the serine protease thrombin. Embryonic chick dorsal root ganglion neurons showed a dramatic depolymerization of actin filaments within 5 min upon hSema III exposure and virtually no influence on microtubules (MT). Only at later time points (20-30 min) was the polymerization/depolymerization rate of MT significantly affected. Thrombin induced a morphologically and kinetically similar growth cone collapse. Moreover, thrombin induced an early and selective depolymerization of dynamic MT, accompanied by the formation of loops of stable MT bundles. Selective changes in the phosphorylation pattern of tau were associated with microtubule assembly in thrombin-induced responses. Our data provide evidence that different signal transduction pathways lead to distinct changes of the growth cone cytoskeleton.

Quantitative estimation of F-actin in single growth cones.

Growth cones at distal ends of elongating neurites are characterized by a bunch of motile filopodia. Filamentous actin (F-actin) is the supporting cytoskeletal structure of growth cone filopodia. Normal growth cone motility requires balanced polymerization and depolymerization rates of F-actin. If this balance is disturbed, growth cone shape is altered and extension may fail. Image acquisition by confocal scanning microscopy was used as a very efficient tool to optically isolate single growth cones from the rest of the cell to study morphological and physiological behavior. The relative F-actin content (r.a.c.) of a single growth cone area was defined as a parameter describing different growth cone states. To estimate r.a.c., a double-labeling technique was applied. F-actin was selectively labeled by fluorescent rhodamine-conjugated phalloidin and total protein was unspecifically labeled by 5-(4, 6-dichlorotriazin-2-yl)aminofluorescein (DTAF). The r.a.c. was calculated by rationing and averaging digitized rhodamine and DTAF fluorescence of single growth cone areas. Subsequently, r.a.c. was used as a numeric descriptor of the variable F-actin underlying morphological structures of growth cones. The method allowed an analysis of local changes in growth cone morphology measured as a change in F-actin due to signaling events. It can be used to quantify ligand-receptor effects at subcellular areas of intact cells.

Bradykinin-induced collapse of rat pheochromocytoma (PC12) cell growth cones: a role for tyrosine kinase activity.

Pathfinding of growing nerve processes is guided by extracellular guidance cues. Here we report growth cone collapse of NGF-differentiated PC12 cells in culture evoked by the neuropeptide bradykinin. The growth cone response is mediated by B2 bradykinin receptors. Two different effects were distinguished. (1) Disappearance of filopodia occurred together with a loss of fibrillar actin (F-actin) in the growth cones at picomolar concentrations of bradykinin. The relative F-actin content was measured by means of rhodamine-phalloidin fluorescence using confocal microscopy. (2) Bradykinin-induced Ca2+ release and retraction of the neurite occurred at nanomolar concentrations. Ca2+ responses at single growth cones were measured using a 1:1 mixture of fura-red and fluo-3 Ca2+-sensitive dyes. The [Ca2+]i rise is not a prerequisite for the observed effects, because F-actin loss and retraction occurred during inhibition of Ca2+ responses. In contrast, inhibition by genistein pointed to a tyrosine kinase activity in the bradykinin-evoked cellular events. Subsequent analysis of phosphotyrosine proteins revealed that bradykinin stimulated tyrosine phosphorylation of the cytoskeleton-associated protein paxillin and the nonreceptor protein tyrosine kinase pp60(c-src). Paxillin and pp60(c-src) co-precipitated after bradykinin treatment. Immunostaining experiments showed punctate distribution of paxillin along PC12 neurites and in growth cones. Taken together, our data suggest that pp60(c-src) and paxillin are putative components of the intracellular signaling pathway of bradykinin-mediated neurite retraction and provide evidence for a crosstalk between G-protein- and tyrosine kinase-dependent pathways in these cellular events.

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites.

Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.

Structure and function of the beta 2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif.

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.

Calyculin-A-induced fast neurite retraction in nerve growth factor-differentiated rat pheochromocytoma (PC12) cells.

Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3-4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca2+ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca2+ level and are most likely due to the inhibition of PP-1 class phosphatases.

Differential modulation of pharmacologically distinct components of Ca2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells.

We have studied the effects of protein kinase C (PKC) activators 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca2+ and K+ channels in nerve-growth-factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba2+ and K+ currents were recorded at room temperature with the patch-clamp technique. By using omega-conotoxin (CgTX) and isradipine, two specific Ca2+ channel blockers, we found three types of Ba2+ currents (IBa): (1) a omega-CgTX-sensitive IBa; (2) an isradipine-sensitive IBa; and (3) a omega-CgTX plus isradipine-resistant IBa. The external application of 4 beta-PMA or OAG down-modulated the isradipine-sensitive IBa whereas the two other IBa were not affected. 4 beta-PMA-induced inhibition of IBa was prevented by staurosporine (a protein kinase inhibitor) and PKC (19-31) (a specific PKC inhibitor). The delayed rectifier K+ current (IK) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the IBa in different manners. The presence of okadaic acid decreased the isradipine-sensitive IBa more than the omega-CgTX-sensitive IBa. The omega-CgTX plus isradipine-resistant IBa was not affected. Calyculin A down-modulated all three components of IBa to a similar degree. Our results suggest a differential modulation of voltage-activated Ca2+ and K+ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.

Unidirectional interaction between two intracellular calcium stores in rat phaeochromocytoma (PC12) cells.

1. A clone of the rat phaeochromocytoma cell line (PC12) was treated with nerve growth factor (NGF) for 4-6 days and used to study caffeine- and bradykinin-induced Ca2+ release from intracellular Ca2+ stores. The caffeine-sensitive store can be depleted by Ca(2+)-induced Ca2+ release (CICR), while the bradykinin-induced release is mediated by inositol 1,4,5-trisphosphate (IP3). The effect of Ca2+ release from these Ca2+ stores on cytosolic free Ca2+ ([Ca2+]i) was measured by means of fura-2 single cell microfluorimetry. 2. Caffeine application caused no or only a small Ca2+ release in untreated cells in normal culture medium. The caffeine-sensitive pool could be filled by Ca2+ entry into cells through either voltage-activated Ca2+ channels or ligand-gated cation channels. 3. Bradykinin application produced substantial Ca2+ release in untreated cells in normal culture medium. The response was enhanced after K(+)-depolarization of the cells. The bradykinin-induced release of Ca2+ also caused depletion of the caffeine-sensitive pool by CICR. However, Ca2+ released from the IP3-sensitive store was not sequestered into the caffeine-sensitive Ca2+ store. 4. The caffeine-induced rise in [Ca2+]i was blocked by ryanodine in a use-dependent manner. In addition, a substantial use-dependent ryanodine block resulted from the bradykinin-induced rise of [Ca2+]i and subsequent CICR. By contrast, the K(+)-induced rise of [Ca2+]i caused only a marginal use-dependent ryanodine inhibition of Ca2+ release. 5. Our results suggest an enhancement of the IP3-induced [Ca2+]i rise in the cytoplasm by CICR from the caffeine-sensitive pool. 6. A mathematical model adequately simulates our experimental data.

Inhibition of protein kinases in rat pheochromocytoma (PC12) cells promotes morphological differentiation and down-regulates ion channel expression.

We have studied morphological differentiation and ion channel expression in PC12 cells under different culture conditions. Differentiation mediated by nerve growth factor (NGF) was compared with that induced by depletion and inhibition of protein kinases (phorbol ester beta-PMA plus staurosporine). Morphological differentiation was similar under both conditions. However, ion channel densities, studied by means of the patch-clamp technique, were enhanced by NGF and reduced by beta-PMA+staurosporine. Similar changes were also observed for omega-conotoxin-sensitive Ca2+ channels by measuring radioligand binding. The decrease in Ca2+ channel density, after treatment of the cells with beta-PMA+staurosporine, resulted in a reduced increase in the intracellular Ca2+ concentration during K+ depolarization. We conclude that morphological differentiation, but not ion channel expression, can occur during depression of protein kinase activities in PC12 cells.

Inhibition of membrane currents and rises of intracellular Ca2+ in PC12 cells by CGS 9343B, a calmodulin inhibitor.

The calmodulin inhibitor 1,3-dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin - 4-yl)methyl)-4-piperidinyl]-2H-benzimidazol-2-one maleate (CGS 9343B) caused a reversible block of voltage-activated Ca2+, Na+, and K+ currents in differentiated rat pheochromocytoma (PC12) cells. The drug also inhibited nicotinic acetylcholine receptor (nAChR) channel currents but not inward currents evoked by extracellular ATP. Depolarization-induced intracellular Ca2+ transients were almost completely inhibited in growth cones and cell bodies by CGS 9343B. Our results suggest actions of CGS 9343B on ion fluxes unrelated to calmodulin inhibition.

Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel.

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.

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells.

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.

Regulation of bradykinin- and ATP-activated Ca(2+)-permeable channels in rat pheochromocytoma (PC12) cells.

Membrane currents activated by bradykinin (500 nM) and by extracellular ATP (50 microM) were studied in voltage-clamped, NGF-treated rat pheochromocytoma (PC12) cells. Under quasiphysiological ionic conditions, both substances caused an outward current due to opening of Ca(2+)-activated K+ channels. Bradykinin caused an additional inward current that could be studied after blockade by internal Cs+ of the initial transient outward current. The inward current became larger when the extracellular Ca2+ concentration was increased. Neither inositol-1,4,5-trisphosphate, dioctanoylglycerol, phorbol 12-myristat 13-acetate, forskolin, GTP, GTP-gamma-S, or pretreatment with pertussis toxin affected this current component. Increasing the internal Ca buffer concentration [EGTA or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid] from 1 to 10 mM had no effect on the inward current as long as the free [Ca2+]i was kept constant. However, it was modulated by the resting free [Ca2+]i. Elevation of [Ca2+]i from nominally 0 to 60 or to 180 nM increased the bradykinin-induced average peak current density from 0.14 to 1.04 or to 2.29 pA/pF, respectively. This regulation may depend on a calmodulin-dependent pathway, since CGS 9343B, a calmodulin inhibitor, blocked the effect of elevated [Ca2+]i. With ATP as an agonist, outward current was preceded by a large inward current that was partially blocked by extracellular Ca2+ in the millimolar range. Extracellular Ca2+ was also found to reduce the single-channel conductance estimated from outside-out patches treated with ATP.

Dependence of cytosolic calcium in differentiating rat pheochromocytoma cells on calcium channels and intracellular stores.

1. The rat clonal pheochromocytoma cell line (PC12) was used to study changes in the free intracellular Ca2+ concentration [( Ca2+]i) that are related to the distribution of L-type (dihydropyridine-sensitive) and N-type (omega-conotoxin-sensitive) calcium channels during nerve growth factor (NGF)-induced outgrowth of neurites. Changes in [Ca2+]i during K+ depolarization were recorded by means of Fura-2 single-cell microfluorimetry. 2. The basal [Ca2+]i of cells at rest was not altered by long-term treatment with NGF, neither in the cell bodies nor in the growth cones. K+ depolarization of the cells caused a rise in [Ca2+]i. 3. The dihydropyridine (DHP) nifedipine alone, or together with omega-conotoxin (omega-CgTX), were similarly effective in inhibiting the K(+)-induced increase in [Ca2+]i in untreated and NGF-treated cell bodies, arguing for a preferential distribution of L-type Ca2+ channels in this cell area. By contrast, after 6-7 days exposure to NGF the K(+)-induced initial transient rise of [Ca2+]i in growth cones was very sensitive to omega-CgTX, whereas nifedipine affected only the sustained rise. 4. PC12 cells also contain caffeine- and inositol trisphosphate (IP3)-sensitive intracellular Ca2+ stores. Addition of 30 mM-caffeine caused a fast transient rise in [Ca2+]i. The extent of filling of the caffeine-sensitive pool affected basal [Ca2+]i. These Ca2+ storage sites were empty under normal culture conditions. However, a single K+ depolarization caused filling of the stores, followed by spontaneous depletion (50% in about 5 min) after wash-out of high [K+]o. When the caffeine-sensitive stores were empty, the rise in [Ca2+]i was attenuated during submaximal depolarization. Caffeine-sensitive Ca2+ stores were also present in some growth cones, though with much smaller capacities than in cell bodies. 5. Mobilization of Ca2+ from the IP3-sensitive store, by bradykinin exposure, was found to be independent of the caffeine-sensitive pool. There was no apparent 'cross-talk' between both Ca2+ pools. 6. We conclude that changes in [Ca2+]i in cell bodies depend on both membrane Ca2+ channels and intracellular Ca2+ stores. During NGF-induced differentiation there is a predominance of N-type Ca2+ channels in growth cones, while Ca2+ stores are of minor importance in these structures.

Depolarization-induced changes of free intracellular Ca(2+) concentration and of [(3)H]dopamine release in undifferentiated and differentiated PC12 cells.

The rat pheochromocytoma cell line PC12 undergoes morphological differentiation into neurone-like cells when exposed to nerve growth factor (NGF). We found that in PC12 cells the enzymatic activity of choline-acetyltransferase and of tyrosine hydroxylase changed very little during NGF exposure, while tyrosine hydroxylase activity increased during other treatments of the cells. These enzymes are rate limiting for the synthesis of cholinergic and adrenergic neurotransmitters, respectively. In order to learn more about mechanisms involved in catecholamine release from differentiating PC12 cells, we studied the effects of dihydropyridines and of ?-conotoxin on K(+)-depolarization-induced [Ca(2+)](i) transients measured with fura-2 and on [(3)H]dopamine release. These drugs act on different types of voltage-gated Ca channels. We found that in differentiated cells [Ca(2+)](i) transients in growth cones are more sensitive to ?-conotoxin than to dihydropyridines, while the opposite is true for cell bodies in both differentiated and undifferentiated cells. Concomitantly we saw a shift from dihydropyridine-sensitive [(3)H]dopamine release in undifferentiated cells to ?-conotoxin-sensitive release in differentiated cells. We conclude that different types of Ca channels are predominantly involved in [(3)H]dopamine release in undifferentiated and differentiated PC12 cells, respectively.

Hydrophobic properties of the beta 1 and beta 2 subunits of the rat brain sodium channel.

Voltage-sensitive sodium channels purified from rat brain in functional form consist of a stoichiometric complex of three glycoprotein subunits, alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. The hydrophobic properties of these three subunits were examined by covalent labeling with the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which labels transmembrane segments in integral membrane proteins. All three subunits of the sodium channel were labeled by [125I]TID when the purified protein was solubilized in mixed micelles of Triton X-100 and phosphatidylcholine (4:1). The half-time for photolabeling was approximately 7 min consistent with the half-time of 9 min for photolysis of TID under our conditions. Comparable amounts of TID per mg of protein were incorporated into each subunit. Purified sodium channels reconstituted in phosphatidylcholine vesicles were also labeled by TID with comparable incorporation per mg of protein into all three subunits. The efficiency of photolabeling of the three subunits was reduced from 39 to 44% by a 2-fold expansion of the hydrophobic phase of the reaction mixture but was unaffected by a 2-fold expansion of the aqueous phase, confirming that the photolabeling reaction took place in the lipid phase of the vesicle bilayer. The hydrophobic properties of the sodium channel subunits were examined further using phase separation in the nonionic detergent Triton X-114. Under conditions in which beta 1 is dissociated from alpha, the beta 1 subunit was preferentially extracted into the Triton X-114 phase, and the disulfide-linked alpha beta 2 complex was retained in the aqueous phase. When the disulfide bonds between the alpha and beta 2 subunits were reduced with dithioerythritol, the beta 2 subunit was also preferentially extracted into the Triton X-100 phase leaving the free alpha subunit in the aqueous phase. A preparative method for isolation of the beta 1 and beta 2 subunits was developed based on this technique. Considered together, the results of our hydrophobic labeling and phase separation experiments indicate that the alpha, beta 1, and beta 2 subunits all have substantial hydrophobic domains that may interact with the hydrocarbon phase of phospholipid bilayer membranes. Since the alpha subunit is known to be a transmembrane protein with many potential membrane-spanning segments, we conclude that the beta 1 and beta 2 subunits are likely to also be integral membrane proteins with one or more membrane-spanning segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle.

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: alpha (167 kDa), beta (54 kDa), and gamma (30 kDa). Cleavage of disulfide bonds reveals two distinct alpha polypeptides and an additional component, delta. The alpha 1 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). alpha 2, a 143-kDa glycoprotein, has none of the properties characteristic of alpha 1 but binds lectins and contains about 25% N-linked carbohydrate. alpha 2 is disulfide-linked to delta, a 24- to 27-kDa glycopeptide. beta (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. gamma (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purified anti-alpha 1 antibodies or alpha 2-specific lentil lectin-agarose demonstrated that alpha 1 alpha 2 beta gamma delta behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the alpha 2 delta complex dissociates from alpha 1 beta gamma in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.