Rapid and Sensitive Single-Step Radiochemical Assay for Catechol-O-Methyltransferase

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol- O -methyltransferase activity is described. The method is based on the conversion of catechol to [³H]guaiacol by catechol- O -methyltransferase in the presence of Mg²⁺, adenosine deaminase and S -adenosyl l -[methyl-³H]methionine. Incubation and direct extraction of [³H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol- O -methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.     

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min⁻¹/g⁻¹ cells) were achieved at an O₂ delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO₂ and inactivation of catechol-2,3-dioxygenase at high dO₂ levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.     

Initial steps of phloroglucinol metabolism in Penicillium simplicissimum

The pathway for the aerobic catabolism of 1,3,5-trihydroxybenzene (phloroglucinol) by a new strain of Penicillium was investigated using both in vivo and in vitro cell-free systems. The fungal strain was isolated by enrichment on phloroglucinol and identified as P. simplicissimum (Oud) Thom. It grew optimally at pH 5.5 and 27°C with 119 mM (1.5%w/v) of phloroglucinol in a basal mineral salts medium. Vapours of the crystalline substrate placed in a Petri-plate lid supported the growth of the fungal colonies on the agar surface. Mycelia grown on phloroglucinol accumulated 1,2,4-trihydroxybenzene and resorcinol in the medium. Washed, resting mycelia grown on phloroglucinol, when resuspended in a buffer utilized oxygen in the presence of catechol, resorcinol, pyrogallol and phloroglucinol. A NADPH-dependent reductase in the cell-free extract reduced phloroglucinol to dihydrophloroglucinol. This electron donor could not be replaced by NADH. Resorcinol hydroxylase, phloroglucinol reductase, catechol-1,2-oxygenase, and catechol-2,3-oxygenase were detected in cell-free extracts of mycelia grown on phloroglucinol. The possible steps in the degradation of phloroglucinol are discussed.     

Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus n657

The TOL plasmid-encoded enzymes of the methyl-benzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate. Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates. The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol. The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate. Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents. Electronegative substituents strongly inhibited catechol cleavage. The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.     

Ethanol-Induced Oxidative Stress: The Role of Binaphthyl Diselenide as a Potent Antioxidant

It is widely accepted that oxidative stress plays a central role in alcohol-induced pathogenesis. The protective effect of binaphthyl diselenide (NapSe)2 was investigated in ethanol (Etoh)-induced brain injury. Thirty male adult Wistar rats were divided randomly into five groups of six animals each and treated as follows: (1) The control group received the vehicle (soy bean oil, 1 mL/kg, p.o.). (2) Ethanol group of animals was administered with ethanol (70% v/v, 2 mL/kg, p.o.). (3) (NapSe)2 1 mg/kg, 1 mL/kg plus ethanol 70% (v/v, 2 mL/kg, p.o. (5) (NapSe)2 10 mg/kg, 1 mL/kg) plus ethanol 70% (v/v, 2 mL/kg, p.o). After acute treatment, all rats were sacrificed by decapitation. Evidence for oxidative stress in rat brain was obtained from the observed levels of thiobarbituric acid reactive species, of non-protein thiol (NPSH) groups, and of ascorbic acid, as well as from the activities of catalase (CAT) and of superoxide dismutase (SOD). (NapSe)2 compensated the deficits in the antioxidant defense mechanisms (CAT, SOD, NPSH, and ascorbic acid), and suppressed lipid peroxidation in rat brain resulting from Etoh administration. It was concluded that ethanol exposure causes alterations in the antioxidant defense system and induces oxidative stress in rat brain. (NaPSe)2 at 5 mg/kg restored the antioxidant defenses in rat brain and mitigated the toxic effects of alcohol, suggesting that could be used as a potential therapeutic agent for alcohol-induced oxidative damage in rat brain.     

Determination of acteoside in Cistanche deserticola and Boschniakia rossica and its pharmacokinetics in freely-moving rats using LC-MS/MS

A sensitive LC-MS/MS method with a simple solid-phase extraction for the determination of acteoside in rat plasma and tissue homogenates was established for the investigation of bioavailability and brain distribution in freely-moving rats. Acteoside in Cistanche deserticola and Boschniakia rossica was also determined. Acteoside and internal standard were separated on a RP-select B column (125mmx4.6mm i.d., particle size 5microm). The mobile phase consisted of 35% methanol and 65% acetic acid-water (1:100, v/v) at a flow-rate of 1mL/min. Acteoside and the internal standard were monitored using the multiple-reaction monitoring (MRM) mode at m/z transitions of 623-->161 and 609-->301, respectively. The acteoside content was 38.4+/-2.4mg/kg (n=3) for B. rossica, which is obviously lower than 21134.2+/-805.5mg/kg (n=3) of C. deserticola. The protein binding in rat plasma was 75.5+/-1.8%. The brain distribution result indicated that acteoside was evenly distributed in brain tissues (brain stem, cerebellum, the rest of the brain, cortex, hippocampus and striatum) which was about 0.45-0.68% of that in plasma (4.5+/-0.5microg/mL) after 15min of acteoside administration (10mg/kg, i.v.). After acteoside was given (3mg/kg, i.v.; 100mg/kg, p.o.), the oral bioavailability (AUC(p.o.)/dose(p.o.))/(AUC(i.v.)/dose(i.v.)) was only 0.12%.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

On the Metabolism of [3H]Noradrenaline in Different Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were pretreated with either reserpine or desmethylimipramine, either alone or in combination with tropolone. At either 10 min or 1 h after the intraventricular injection of [³H]noradrenaline, in several brain regions the complete metabolic patterns were determined: normetanephrine; the glycol metabolites (methylated and nonmethylated) and their sulfate conjugates; and the acidic metabolites (methylated and nonmethylated). A reserpine-induced increase in the turnover of [³H]noradrenaline caused a transient increase of the catechol glycol followed by elevated levels of the two glycol sulfates. The stimulated [³H]noradrenaline turnover if achieved by desmethylimipramine caused a transient increase of normetanephrine and initially lowered values of catechol glycols (both free and sulfated), which were followed by elevated levels. Drug-pretreated rats compensated for the inhibition of catechol- O -methyltransferase by tropolone in different ways: Reserpine caused an early increase of the catechol glycol beyond the measurements in other treatment groups, whereas desmethylimipramine increased the nonmethylated carboxylic acid and glycol sulfates rather slowly to levels beyond those of other groups. The results support the existence of two compartments with a fast metabolism (an intraneuronal monoamine oxidase compartment and an extraneuronal catechol- O -methyltransferase compartment). In addition, there seems to exist another extraneuronal space with a slow, monoamine oxidase-dependent noradrenaline turnover.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.     

Genetic variation in COMT and PRODH is associated with brain anatomy in patients with schizophrenia

Haploinsufficiency of 22q11 genes including catechol- O -methyltransferase (COMT) and proline dehydrogenase (PRODH) may result in structural and functional brain abnormalities and increased vulnerability to schizophrenia as observed in patients with microdeletions of 22q11. Thus, COMT and PRODH could be modifier genes for schizophrenia. We examined association of polymorphisms in COMT and PRODH with brain anatomy in young patients with schizophrenia and schizoaffective disorder. We acquired structural magnetic resonance imaging data from 51 male patients and genotyped two single nucleotide polymorphisms (SNPs) in the COMT gene and three in the PRODH gene. Statistical Parametric Mapping software and optimized voxel-based morphometry were used to determine regional gray matter (GM) and white matter (WM) density differences, and total GM and WM volume differences between genotype groups. Two nonsynonymous SNPs in the PRODH gene were associated with bilateral frontal WM density reductions and an SNP in the P2 promoter region of COMT (rs2097603) was associated with GM increase in the right superior temporal gyrus. Furthermore, we found evidence for COMT and PRODH epistasis: in patients with a COMT Val allele (rs4680) and with one or two mutated PRODH alleles, we observed increased WM density in the left inferior frontal lobe. Our results suggest that genetic variation in COMT and PRODH has significant effects on brain regions known to be affected in schizophrenia. Further research is needed to investigate the role of 22q11 genes on brain structure and function and their role in vulnerability for schizophrenia.     

Contribution of Sulfate Conjugation, Deamination, and O-Methylation to Metabolism of Dopamine and Norepinephrine in Human Brain

Abstract: The kinetic constants were determined for dopamine (DA) and norepinephrine (NE) metabolism by phenolsulfotransferase (PST), type A and B monoamine oxidase (MAO), and membrane-bound and soluble catechol- O - methyltransferase (COMT) in frontal lobe preparations of human brain. PST and membrane-bound COMT were found to have the lowest K _m, values for both catecholamines. By means of the appropriate rate equations and the calculated kinetic constants for each enzyme, the activity of each enzymatic pathway was determined at varying concentrations of DA and NE. Results indicate that deamination by MAO is the principal pathway for the enzymatic inactivation of DA whereas NE is largely metabolized by MAO type A and membrane-bound COMT under the in vitro assay conditions used. At concentrations less than 100 μ M , soluble COMT'contributes less than 5% to the total catabolism of either catecholamine. PST can contribute up to 15% of the total DA metabolism and 7% of NE metabolism.     

Bacterial catabolism of sulfanilic acid via catechol-4-sulfonic acid

Abstract A sulfanilic acid (4-aminobenzenesulfonic acid) degrading culture consisting of two strains (strain S1 and S2), was studied. Only strain S1 was able to attack sulfanilic acid. When strain S1 was cultavated in a mineral medium with sulfanilic acid an intensive violet colour was observed. The accumulating metabolite was isolated from the culture supernatant. By comparison with an authentic compound the metabolite was identified as catechol-4-sulfonic acid by thin layer and high performance liquid chromatography and by UV- and H-NMR spectroscopy. The occurrence of catechol-4-sulfonic acid indicates that there is no release of the sulfonic group before ring cleavage.     

THE EFFECTS OF METOPIRONE AND ADRENALECTOMY ON THE REGULATION OF THE ENZYMES MONOAMINE OXIDASE AND CATECHOL-O-METHYL TRANSFERASE IN DIFFERENT BRAIN REGIONS

Abstract— The role of glucocorticoids in the regulation of the enzymes monoamine oxidase (MAO) and catechol- O -methyltransferase (COMT) in brain regions has been studied. Glucocorticoids were blocked by Metopirone. The activities of MAO and COMT were determined in the hypophysis, hypothalamus, pineal gland and in the rest of brain. All the cerebral tissues except the pineal gland demonstrated highest MAO activity 8 h after Metopirone administration, when glucocorticoids were at the lowest level. Prolonged treatment for 10 days significantly augmented MAO activity in brain, hypophysis and hypothalamus, and COMT in the hypophysis increased by 56 per cent. The COMT activity in the rest of the brain did not change significantly with either short or prolonged administration. Complete ablation of the adrenal cortex resulted in a 167 per cent rise in MAO activity of the hypophysis. Metopirone and hydrocortisone inhibit MAO and COMT in vitro. The results suggest that glucocorticoids in the circulation of normal animals inhibit the activities of MAO and COMT. The inhibition or ablation of these hormones removes this rate-limiting control of catecholamine degradation resulting in higher activities of MAO and COMT. Metopirone, an inhibitor of MAO and COMT in vitro , acts in the opposite direction in vivo due to its inhibitory effects on corticoid biosynthesis.     

Sulfation of Dopamine and Other Biogenic Amines by Human Brain Phenol Sulfotransferase

Abstract: Phenol sulfotransferase was isolated in 100,000g supernatant fractions prepared from postmortem samples of human brain. Since phenol sulfotransferase (PST) has been shown to conjugate the amine neurotransmit-ters in vivo , the abilities of eight different biogenic amines and structurally related compounds to act as substrates for PST were studied. These experiments demonstrate that at a concentration of 20 μM, dopamine (DA) was the best substrate examined and was followed in decreasing order of activity by 3-methoxytyramine (3-MT), tyramine, norepinephrine, 3-methoxy-4-hydroxyphenylethyleneglycol, octopamine, 5-hydroxytryptamine and dihydroxyphenylethyleneglycol. At a substrate concentration of 100 /UM the relative order of activity was altered, so that tyramine became the most rapidly conjugated substrate while the activity of DA and 3-MT relative to the other substrates tested was diminished. This change in substrate affinity with differing substrate concentrations can be explained, at least for DA, by the occurrence of apparent substrate inhibition at concentrations above 25 to 30 μM. Using PST isolated in 100,000g supernatant fractions from human brain, the K_m value for DA was found to be 5.0 μM, while the K_m value for the sulfate-donor 3'-phosphoadenosine-5'-phosphosulfate was 0.25 μM. The ratio of 3- O - to 4- O -DA-sulfate formed in vitro by human brain PST was found to be about 4: 1. In addition, both the 3- O - and 4- O -esters were found not to be deaminated by human brain mitochondrial MAO. The relative role of PST with respect to MAO and catechol- O -methyltransferase in the degradation of the biogenic amine neurotransmitters in human brain is discussed.     

A Rapid and Simple Method for the Determination of Picogram Levels of 3-Methoxytyramine in Brain Tissue Using Liquid Chromatography with Electrochemical Detection

Abstract: A rapid and simple technique using solvent extraction, ion-pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3-methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3-methoxytyramine added to the samples is 45–50%. 3-Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3-methoxytyramine levels in rat striata, while catechol- O -methyltransferase inhibition with 3',4'-dihydroxy-2 methylpropiophenone completely depleted 3-methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D-amphetamine were also examined. The 3-methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.     

Distinct Cellular Localization of Membrane-Bound and Soluble Forms of Catechol-O-Methyltransferase in Brain

Abstract: The cellular localization of the two forms of catechol- O -methyltransferase (COMT) was investigated by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid, which causes degeneration of striatal neurons followed by proliferation of astroglial cells. Membrane-bound COMT activity was decreased in the lesioned striatum, while soluble COMT activity was increased. There was a statistically significant correlation between the ratio of lesioned to control activity for membrane-bound COMT and the neuronal marker enzyme glutamate decarboxylase. Similarly the increase in soluble COMT activity paralleled that of the astroglial marker enzyme, glutamine synthetase. These results indicate that the K _m membrane-bound catechol- O -methyltransferase may be localized predominantly in neurons, whereas the high-K_m soluble enzyme is found in glial cells.     

A sensitive radioenzymatic assay for the simultaneous determination of salsolinol and dopamine

A sensitive radioenzymatic assay for the simultaneous quantitation of salsolinol and dopamine in tissues and fluids has been developed. Salsolinol and dopamine were radiolabeled by 0-methylation using the enzyme catechol-0-methyltransferase and its cosubstrate, [3H]-S-adenosylmethionine, as the methyl donor. Specificity was achieved by alumina adsorption, selective solvent extraction, thin layer chromatography, primary amine precipitation and ion pair solvent extraction. The assay was linear over a 1000 fold concentration range. Sensitivities of 2 and 3 picograms were obtained for dopamine and salsolinol, respectively. Separate assay of standard samples had a coefficient of variation of 5%. Salsolinol was formed $\text{in vitro}$ in dopamine enriched plasma and whole brain homogenates following incubation with physiologic concentrations of acetaldehyde.     

Phosphorylated dolichols in aging.

The age-associated changes in the levels and synthesis of dolichyl phosphate and dolichyl diphosphate derivatives were investigated in brain and liver of 057B1/NNia mice. The total chloroform/methanol (2:1, v/v)-extractable phosphorylated dolichols of brain increased from 1.01 micrograms/g at 3 months to 5.22 micrograms/g at 28 months of age. The long-chain dolichyl diphosphate oligosaccharide (Dol-PP-oligo) levels of brain increased from 0.82 microgram/g in 3 months to 2.8 micrograms/g in 28-month-old animals. However, in liver and in kidney, the levels of these components were unaffected by age. Incorporation of labelled glucose from UDP-glucose into dolichyl phosphate glucose and Dol-PP-oligo in brain microsomes was unaffected by age, whereas, in liver microsomes, the rates of synthesis of both components increased by 50-150%. The increased rate of synthesis and lack of accumulation of Dol-PP-oligo in liver suggest an active utilization and/or catabolism of these glycoprotein precursors. The accumulation of Dol-PP-oligo in aging brain may reflect its decreased utilization for N-glycosylation and/or reduced catabolism.     

Regulation of maximal open probability is a separable function of Ca(v)beta subunit in L-type Ca2+ channel, dependent on NH2 terminus of alpha1C (Ca(v)1.2alpha)

beta subunits (Ca(v)beta) increase macroscopic currents of voltage-dependent Ca2+ channels (VDCC) by increasing surface expression and modulating their gating, causing a leftward shift in conductance-voltage (G-V) curve and increasing the maximal open probability, P(o,max). In L-type Ca(v)1.2 channels, the Ca(v)beta-induced increase in macroscopic current crucially depends on the initial segment of the cytosolic NH2 terminus (NT) of the Ca(v)1.2alpha (alpha1C) subunit. This segment, which we term the "NT inhibitory (NTI) module," potently inhibits long-NT (cardiac) isoform of alpha1C that features an initial segment of 46 amino acid residues (aa); removal of NTI module greatly increases macroscopic currents. It is not known whether an NTI module exists in the short-NT (smooth muscle/brain type) alpha(1C) isoform with a 16-aa initial segment. We addressed this question, and the molecular mechanism of NTI module action, by expressing subunits of Ca(v)1.2 in Xenopus oocytes. NT deletions and chimeras identified aa 1-20 of the long-NT as necessary and sufficient to perform NTI module functions. Coexpression of beta2b subunit reproducibly modulated function and surface expression of alpha1C, despite the presence of measurable amounts of an endogenous Ca(v)beta in Xenopus oocytes. Coexpressed beta2b increased surface expression of alpha1C approximately twofold (as demonstrated by two independent immunohistochemical methods), shifted the G-V curve by approximately 14 mV, and increased P(o,max) 2.8-3.8-fold. Neither the surface expression of the channel without Ca(v)beta nor beta2b-induced increase in surface expression or the shift in G-V curve depended on the presence of the NTI module. In contrast, the increase in P(o,max) was completely absent in the short-NT isoform and in mutants of long-NT alpha1C lacking the NTI module. We conclude that regulation of P(o,max) is a discrete, separable function of Ca(v)beta. In Ca(v)1.2, this action of Ca(v)beta depends on NT of alpha1C and is alpha1C isoform specific.