RESUMO
The objective of this study was to identify through quantum mechanical quantitative structure activity relationships (Q-QSARs) chemical structures in dental monomers that influence their mutagenicity. AMPAC, a semiempirical computer program that provides quantum mechanical information for chemical structures, was applied to three series of reference chemicals: a set of methacrylates, a set of aromatic and a set of aliphatic epoxy compounds. QSAR models were developed using this chemical information together with mutagenicity data (Salmonella TA 100, Ames Test). CODESSA, a QSAR program that calculates quantum chemical descriptors from information generated by AMPAC and statistically matches these descriptors with observed biological properties was used. QSARs were developed which had r2 values exceeding 0.90 for each study series. These QSARs were used to accurately predict the mutagenicity of BISGMA. a monomer commonly used in dentistry, and two epoxy monomers with developing use in dentistry, GY-281 and UVR-6105. The Q-QSAR quantum mechanical descriptors correctly predicted the level of mutagenicity for all three compounds. The descriptors in the correlation equation pointed to components of structure that may contribute to mutagenesis. The QSARs also provided 'dose windows' for testing mutagenicity, circumventing the need for extensive dose exploration in the laboratory. The Q-QSAR method promises an approach for biomaterials scientists to predict and avoid mutagenicity from the chemicals used in new biomaterial designs.
Assuntos
Esmalte Dentário/química , Mutagênicos , Relação Dose-Resposta a Droga , Metacrilatos/química , Modelos Químicos , Testes de Mutagenicidade , Teoria Quântica , Software , Relação Estrutura-AtividadeRESUMO
Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca(2+) channels and as voltage sensors for coupling membrane depolarization to release of Ca(2+) from the sarcoplasmic reticulum. In skeletal muscle, the principal or alpha(1S) subunit occurs in full-length ( approximately 10% of total) and post-transcriptionally truncated ( approximately 90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized alpha(1S) subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (alpha(1S)) or truncated (alpha(1SDeltaC)) alpha subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Q(max) 80-150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in alpha(1S), unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba(2+) currents were small relative to Q(max). The truncated alpha(1SDeltaC) protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the alpha(1S) subunit conduct the majority of the L-type Ca(2+) current in skeletal muscle. Our data also suggest that the carboxyl terminus of the alpha(1S) subunit modulates the coupling between charge movement and channel opening.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Animais , Canais de Cálcio Tipo L/genética , DNA Complementar/genética , Feminino , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Músculo Esquelético/metabolismo , Oócitos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
1. A truncated form of the rabbit alpha1S Ca2+ channel subunit (alpha1SDeltaC) was expressed with the beta1b, alpha2delta and gamma auxiliary subunits in Xenopus laevis oocytes. After 5-7 days, skeletal muscle L-type currents were measured (469 +/- 48 nA in 10 mM Ba2+). All three of the auxiliary subunits were necessary to record significant L-type current. A rapidly inactivating, dihydropyridine-insensitive endogenous Ba2+ current was observed in oocytes expressing the auxiliary subunits without an exogenous alpha subunit. Expression of full-length alpha1S gave 10-fold smaller currents than the truncated form. 2. Three missense mutations causing hypokalaemic periodic paralysis (R528H in domain II S4 of the alpha1S subunit; R1239H and R1239G in domain IV S4) were introduced into alpha1SDeltaC and expressed in oocytes. L-type current was separated from the endogenous current by nimodipine subtraction. All three of the mutations reduced L-type current amplitude ( approximately 40 % for R528H, approximately 60-70 % for R1239H and R1239G). 3. The disease mutations altered the activation properties of L-type current. R528H shifted the G(V) curve approximately 5 mV to the left and modestly reduced the voltage dependence of the activation time constant, tauact. R1239H and R1239G shifted the G(V) curve approximately 5-10 mV to the right and dramatically slowed tauact at depolarized test potentials. 4. The voltage dependence of steady-state inactivation was not significantly altered by any of the disease mutations. 5. Wild-type and mutant L-type currents were also measured in the presence of (-)-Bay K8644, which boosted the amplitude approximately 5- to 7-fold. The effects of the mutations on the position of the G(V) curve and the voltage dependence of tauact were essentially the same as in the absence of agonist. Bay K-enhanced tail currents were slowed by R528H and accelerated by R1239H and R1239G. 6. We conclude that the domain IV mutations R1239H and R1239G have similar effects on the gating properties of the skeletal muscle L-type Ca2+ channel expressed in Xenopus oocytes, while the domain II mutation R528H has distinct effects. This result implies that the location of the substitutions is more important than their degree of conservation in determining their biophysical consequences.
Assuntos
Canais de Cálcio Tipo L/genética , Músculo Esquelético/metabolismo , Mutação , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Bário/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Expressão Gênica , Paralisia Periódica Hipopotassêmica/genética , Nimodipina/farmacologia , Oócitos , Técnicas de Patch-Clamp , RNA Complementar , Coelhos , Xenopus laevisRESUMO
The pore of the catfish olfactory cyclic nucleotide-gated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (approximately 7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted.
Assuntos
Ácidos Carboxílicos/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Nucleotídeos Cíclicos/fisiologia , Prótons , Animais , Sítios de Ligação/fisiologia , Peixes-Gato , Dimerização , Condutividade Elétrica , Glutamatos/metabolismo , Hibridização Genética , Concentração de Íons de Hidrogênio , Canais Iônicos/química , Canais Iônicos/genética , Mutação , Oócitos , Técnicas de Patch-Clamp , Permeabilidade , Isoformas de Proteínas/metabolismo , Xenopus laevisRESUMO
The skeletal muscle L-type Ca channel serves a dual role as a calcium-conducting pore and as the voltage sensor coupling t-tubule depolarization to calcium release from the sarcoplasmic reticulum. Mutations in this channel cause hypokalemic periodic paralysis (HypoPP), a human autosomal dominant disorder characterized by episodic failure of muscle excitability that occurs in association with a decrease in serum potassium. The voltage-dependent gating of L-type Ca channels was characterized by recording whole-cell Ca currents in myotubes cultured from three normal individuals and from a patient carrying the HypoPP mutation R528H. We found two effects of the R528H mutation on the L-type Ca current in HypoPP myotubes: (1) a mild reduction in current density and (2) a significant slowing of the rate of activation. We also measured the voltage dependence of steady-state L-type Ca current inactivation and characterized, for the first time in a mammalian preparation, the kinetics of both entry into and recovery from inactivation over a wide range of voltages. The R528H mutation had no effect on the kinetics or voltage dependence of inactivation.
Assuntos
Canais de Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Músculo Esquelético/metabolismo , Paralisias Periódicas Familiares/metabolismo , Transporte Biológico/fisiologia , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo L , Células Cultivadas , Humanos , Cinética , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Mutação , Paralisias Periódicas Familiares/genética , Fatores de TempoRESUMO
Block of N-type Ca channels by omega-conotoxin GVIA (CgTx) was studied in freshly dissociated bullfrog and rat sympathetic neurons. With 2-5 mM Ba as charge carrier, CgTx blocked almost all of the high-threshold Ca channel current recorded in the presence of nimodipine (3 microM) to block L-type Ca channels. Toxin block reversed slowly (time constant approximately 1 hr) in frog cells and even more slowly in rat cells. CgTx block was faster and more potent in rat cells than frog cells. The rate of block was proportional to CgTx concentration, consistent with 1:1 binding of CgTx to channels. When the external Ba concentration was increased, the development of block was slower, consistent with competition between CgTx and Ba for a binding site. The recovery from block was somewhat faster in higher external Ba. Some cells had significant current remaining in saturating concentrations of nimodipine and CgTx, especially with high Ba concentrations in the external solution. The current resistant to nimodipine and CgTx was activated at lower depolarizations than the CgTx-sensitive current and had faster activation and inactivation kinetics, but unlike low-threshold T-type current, the resistant current had rapidly decaying tail currents.