PubMed · 9002733
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J Frieman. 1996. Alternative protocol.. https://pubmed.ncbi.nlm.nih.gov/9002733/
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1. Whole-cell patch clamp recordings were used to investigate the modulation by reducing and oxidizing agents of recombinant human cardiac L-type Ca2+ channel alpha1C subunits stably expressed in human embryonic kidney (HEK 293) cells. 2. The oxidizing agents thimerosal (10 microM) and p-chloromercuribenzene sulphonic acid (PCMBS; 2 microM to 2 mM) caused irreversible inhibition of Ca2+ channel currents. The reducing agent 1,4-dithiothreitol (DTT; 2 mM) was without effect on Ca2+ channel currents, but reversed the inhibitory actions of thimerosal and PCMBS. 3. Ca2+ channel currents were also inhibited by pretreatment with the methanethiosulphonate compound (2-aminoethyl)methanethiosulphonate (MTSEA, 2.5 mM), but were unaffected by identical pretreatment with (2-sulphonatoethyl)methanethiosulphonate (MTSES, 10 mM). The effects of MTSEA could be fully reversed by DTT (2 mM). The degree of current inhibition caused by 200 microM PCMBS was not significantly affected by pretreatment with MTSEA, and following PCMBS treatment, MTSEA caused a similar degree of inhibition to that observed in cells that were not previously treated with PCMBS. These findings suggested that distinct thiol groups were modulated by these two agents. 4. Hypoxic inhibition of Ca2+ channel currents was unaffected by pretreatment of cells with MTSEA but was fully prevented by treatment with PCMBS. Our results indicate that distinct cysteine residues on the alpha1C subunit can undergo redox modulation and in so doing alter channel function. Some, but not all, of these residues appear to be associated with the mechanism underlying inhibition of this channel by hypoxia.
The three-dimensional structure of omega-conotoxin MVIID has been determined in aqueous solution by two-dimensional 1H NMR techniques. A total of 267 relevant upper-bound distance restraints were used to obtain a family of convergent structures using molecular dynamics methods. A standard simulated annealing protocol using the XPLOR program included in ARIA provided a total of 18 final structures. The averaged RMSD between these structures and the mean atomic coordinates was 0.8 +/- 0.3 A for the backbone atoms. The highest mobility was observed in the segments between residues 10 to 13, comprising Tyr 13, one of the residues shown to be important for binding of omega-conotoxin GVIA and MVIIA to N-type calcium channels. The three-dimensional structure is stabilised by the three disulfide bonds and includes a short antiparallel beta-strand between residues 5-8, 23-25 and 19-21. The folding for this non-N-type calcium channel blocker is similar to that previously calculated for omega-conotoxins GVIA, MVIIA and MVIIC. This suggests the disulfide bond pattern fixes the structure. The reported three-dimensional information can be used to advantage in order to highlight the structural parameters involved in discrimination among calcium channel subtypes.
Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC-MS-MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 microm). The mobile phase consisted of methanol-50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode. The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95+/-2% and 95+/-4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5-100 ng/ml concentration range for the two analytes (r2 = 0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2-4.7% for the metabolite. The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.