Midpoint Of Inactivation Research Articles

Expression defect of the rare variant/Brugada mutation R1512W depends upon the SCN5A splice variant background and can be rescued by mexiletine and the common polymorphism H558R

ABSTRACT Background : Mutations in SCN5A that decrease Na current underlie arrhythmia syndromes such as the Brugada syndrome (BrS). SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss-of-function and rescue for R1512W, a mutation reported to cause BrS. Methods and results : We made the mutation in both variants and expressed them in HEK-293 cells for voltage-clamp study. After 24 hours of transfection, the current expression level of R1512W was reduced by ~50% in both Q1077del and Q1077 compared to the wild-type (WT) channel, respectively. The activation and inactivation midpoint were not different between WT and mutant channels in both splice variant backgrounds. However, slower time constants of recovery and enhanced intermediate inactivation were observed for R1512W/Q1077 compared with WT-Q1077, while the recovery and intermediate inactivation parameters of R1512W/Q1077del were similar to WT-Q1077del. Furthermore, both mexiletine and the common polymorphism H558R restored peak sodium current (I Na) amplitude of the mutant channel by increasing the cell surface expression of SCN5A. Conclusion : These findings provide further evidence that the splice variant affects the molecular phenotype with implications for the clinical phenotype, and they provide insight into the expression defect mechanisms and potential treatment in BrS.

Purification, characterization and specificity of a new GH family 35 galactosidase from Aspergillus awamori

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and β-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50–80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and β-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55–60 °C, 5.0–5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and β-galactosidase activities, but also exhibited β-xylosidase and β-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable β-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.

Cysteine Mutagenesis to Probe Site-Specific Interactions in the Voltage Sensor Module of hNaV1.4

Cysteine scanning mutagenesis was used to probe interactions of positively charged S4 residues with S1-S3 countercharges in domains I, II and IV of the human skeletal muscle sodium channel hNaV1.4. Single cysteine substitution was used to assess the effect of charge neutralization of residues in the voltage sensor module of these domains. Activation and fast inactivation were most affected by cysteine substitution in domains II and IV respectively, while both of these state transitions were affected by like mutations in domain I. Double cysteine mutations in the VSM of each of these domains did not result in disulfide locking. To probe for specific interactions using these mutations, we employed a metal ion bridge approach using cadmium, and assessed its effect on equilibrium parameters of activation and fast inactivation, and peak current amplitude. In domain I, double cysteine mutations of R219C or R222C with the gating charge transfer glutamate (E171C) significantly affected fast inactivation, with activation midpoint significantly depolarized by several DIS4 cysteine / countercharge mutations incorporating the polar residue substitutions N144C and S134C. Domain II double cysteine mutations R672C / E598C and R675C / E598C also significantly depolarized the activation midpoint with cadmium addition. In domain IV, double cysteine mutations R1448C / E1373C and R1448C / N1389C responded to cadmium with slowed entry into fast inactivation, a depolarized inactivation midpoint and a decreased slope. Our results show that certain domain specific functions of the voltage sensor modules in hNaV1.4 occur with pairwise interactions of S4 positive and S1-S3 countercharge residues. This work was supported by NIH R15NS093579-01A1 to JRG and NIH P20GM103408 (INBRE).

KV2.1/KV6.4 Heterotetramers are Functional in Two Stoichiometric Configurations

Members of the voltage-gated K+ (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9, which are collectively designated as electrically silent (KvS) subunits, selectively modulate the biophysical properties of Kv2 channels by forming heterotetrameric Kv2/KvS channel complexes: e.g. Kv2.1/Kv6.4 heterotetramers display a 40 mV hyperpolarizing shift in the voltage-dependence of C-type inactivation. Foster resonance energy transfer (FRET) analysis has previously demonstrated that Kv2.1/Kv9.3 channels are composed of three Kv2.1 subunits and one Kv9.3 subunit. However, it remains unknown whether Kv2.1/KvS channels can have other stoichiometries. We investigated this specifically for the Kv2.1/Kv6.4 combination by comparing the biophysical properties of different concatemeric constructs to these of the corresponding monomers. Kv2.1-Kv2.1 (2_2) dimers yielded delayed rectifier currents with biophysical properties similar to those of channels composed of individual Kv2.1 subunits. Kv2.1-Kv6.4 (2_6) or Kv6.4-Kv2.1 (6_2) dimers displayed a midpoint of inactivation of −59±3 and −58±1 mV (n=6), respectively, similar to that of Kv2.1/Kv6.4 channels obtained from co-transfection the individual subunits (−62±2 mV). This suggested that these channels are functional with a 2:2 stoichiometry. Analysis of tetrameric concatemers revealed that the positional arrangement of the Kv6.4 subunits was crucial. Indeed, we could not detect any functional channels when two Kv6.4 subunits were positioned side by side (2_6_6_2), but the tetramer with alternating subunits (2_6_2_6) displayed a midpoint of inactivation of −62 ±2 mV (n=6). Additionally, tetramers that represented a 3:1 stoichiometry (2_6_2_2) also displayed the hyperpolarizing shift of the inactivation. Interestingly, the voltage-dependence of activation became shallower with incorporation of more Kv6.4 subunits: slope factors were 9 mV for 2_2_2_2, 15 mV for 2_6_2_2 and 22 mV for 2_6_2_6. Taken together, our data suggest that the Kv2.1:Kv6.4 stoichiometry in heteroteramers can be either 3:1 or 2:2 with the restriction that Kv2.1 and Kv6.4 have to alternate in the channel complex in the 2:2 configuration.

P.1.b.008 Association between attenuated brightness mismatch negativity and antipsychotic polytherapy

Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to −40 mV; in full current–voltage curves maximum current occurred between −20 and −10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at −90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 μM. The NaV 1.4 selective toxin μ-CTx-GIIIB reversibly blocked the Na+ current ∼60% at 10 μM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was −72 ± 2 mV (slope factor 7.2 ± 0.2) in automated patch clamp and −74 ± 2 mV (slope factor 7.4 ± 0.4) with conventional recording. The corresponding values for activation mid-point were −33.2 ± 2.4 and −30.3 ± 2.7 mV (slope 5.8 ± 0.3 and 6.4 ± 0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. μ-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels.

Sudden infant death syndrome–associated mutations in the sodium channel beta subunits

Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Effect of magnesium ions on the thermal stability of human poly(A)-specific ribonuclease

Poly(A)-specific ribonuclease (PARN), a member of the DEDD family, is a key enzyme involved in the deadenylation of mRNA in higher eukaryotic cells. In this research, it was found that Mg 2+ could protect PARN against thermal inactivation by increasing the midpoint of inactivation and decreasing the inactivation rate. This protective effect was unique to Mg 2+ in a concentration-dependent manner. However, the thermal unfolding and aggregation was promoted by the addition of Mg 2+ at high temperatures. These results revealed that Mg 2+ might have dual effects on PARN stability: protecting the active site but endangering the overall structural stability.

The influence of protons and zinc ions on the steady-state inactivation of Kv1.3 potassium channels

Using the whole-cell patch-clamp technique, we investigated the influence of extracellular pH and zinc ions (Zn2+) on the steady-state inactivation of Kv1.3 channels expressed in human lymphocytes. The obtained data showed that lowering the extracellular pH from 7.35 to 6.8 shifted the inactivation midpoint (Vi) by 17.4 ± 1.12 mV (n = 6) towards positive membrane potentials. This shift was statistically significant (p < 0.05). Applying 100 μM Zn2+ at pH 6.8 further shifted the Vi value by 16.55 ± 1.80 mV (n = 6) towards positive membrane potentials. This shift was also statistically significant (p < 0.05). The total shift of the Vi by protons and Zn2+ was 33.95 ± 1.90 mV (n = 6), which was significantly higher (p < 0.05) than the shift caused by Zn2+ alone. The Zn2+-induced shift of the Vi at pH 6.8 was almost identical to the shift at pH = 7.35. Thus, the proton-and Zn2+-induced shifts of the Vi value were additive. The steady-state inactivation curves as a function of membrane voltage were compared with the functions of the steady-state activation. The total shift of the steady-state inactivation was almost identical to the total shift of the steady-state activation (32.01 ± 2.10 mV, n = 10). As a result, the “windows” of membrane potentials in which the channels can be active under physiological conditions were also markedly shifted towards positive membrane potentials. The values of membrane voltage and the normalised chord conductance corresponding to the points of intersection of the curves of steady-state activation and inactivation were also calculated. The possible physiological significance of the observed modulatory effects is discussed herein.

Developmental Changes in Two Voltage-Dependent Sodium Currents in Utricular Hair Cells

Two kinds of sodium current (I(Na)) have been separately reported in hair cells of the immature rodent utricle, a vestibular organ. We show that rat utricular hair cells express one or the other current depending on age (between postnatal days 0 and 22, P0-P22), hair cell type (I, II, or immature), and epithelial zone (striola vs. extrastriola). The properties of these two currents, or a mix, can account for descriptions of I(Na) in hair cells from other reports. The patterns of Na channel expression during development suggest a role in establishing the distinct synapses of vestibular hair cells of different type and epithelial zone. All type I hair cells expressed I(Na,1), a TTX-insensitive current with a very negative voltage range of inactivation (midpoint: -94 mV). I(Na,2) was TTX sensitive and had less negative voltage ranges of activation and inactivation (inactivation midpoint: -72 mV). I(Na,1) dominated in the striola at all ages, but current density fell by two-thirds after the first postnatal week. I(Na,2) was expressed by 60% of hair cells in the extrastriola in the first week, then disappeared. In the third week, all type I cells and about half of type II cells had I(Na,1); the remaining cells lacked sodium current. I(Na,1) is probably carried by Na(V)1.5 subunits based on biophysical and pharmacological properties, mRNA expression, and immunoreactivity. Na(V)1.5 was also localized to calyx endings on type I hair cells. Several TTX-sensitive subunits are candidates for I(Na,2).

Properties of voltage-gated Na + channels in the human rhabdomyosarcoma cell-line SJ-RH30: Conventional and automated patch clamp analysis

Conventional and automated patch clamp electrophysiology were used to characterise the Na + current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na + current at potentials positive to −40 mV; in full current–voltage curves maximum current occurred between −20 and −10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at −90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na + current completely at 1 μM. The Na V 1.4 selective toxin μ-CTx-GIIIB reversibly blocked the Na + current ∼60% at 10 μM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was −72 ± 2 mV (slope factor 7.2 ± 0.2) in automated patch clamp and −74 ± 2 mV (slope factor 7.4 ± 0.4) with conventional recording. The corresponding values for activation mid-point were −33.2 ± 2.4 and −30.3 ± 2.7 mV (slope 5.8 ± 0.3 and 6.4 ± 0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na + current. TTX inhibited with an IC 50 of 23 nM. μ-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na + current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant Na V 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na + channels.

A novel molecular inactivation determinant of voltage-gated Ca V1.2 L-type Ca 2+ channel

The inactivation of voltage-gated L-type Ca 2+ channels (Ca V1) regulates Ca 2+ entry and controls intracellular Ca 2+ levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca V1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802–807; poly ED motif) at the II–III loop of the alpha 1 subunits of rabbit of Ca v1.2, α 11.2 and α 11.2 ΔN60–Δ1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca V1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca 2+ or Ba 2+ and was not altered by the β2A subunit. Missing from the fast inactivating neuronal P/Q (Ca V2.1)-, N (Ca V2.2)- or R (Ca V2.3)-type channels and modulating Ca V1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca 2+ channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca V1.2 specific built-in “stopper.” In this model, Ca V1 accomplishes a large Ca 2+ influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.

The Pore, not Cytoplasmic Domains, Underlies Inactivation in a Prokaryotic Sodium Channel

Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from ∼50 ms at depolarized voltages to a maximum of ∼100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by ∼2.5-fold, and the midpoint of the steady-state inactivation curve was shifted ∼20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.

High-voltage-activated calcium channels in Muller cells acutely isolated from tiger salamander retina.

Muller cells mediate retinal function by stabilizing the ionic environment and signal glial network activity via calcium waves. Using whole-cell patch clamp recording, we describe a high-voltage-activated, slowly inactivating Ca channel current in isolated salamander Muller cells that has unusual pharmacological properties. The Ca channel current has an activation midpoint of approximately -8 mV and an inactivation midpoint of approximately -26 mV in 10 mM Ba2+. The time constant for inactivation is approximately 380 ms at potentials positive to zero. The current is blocked by Cd2+ with an EC50 of <100 nM. nisoldipine (10 microM) blocks approximately 50%, while nifedipine (1 microM), diltiazem (20 microM), and verapamil (50 microM) each block one-third of the current. In contrast to its typical actions, BayK 8644 blocks the current by approximately 25%. Blockers of other Ca channel subtypes were also tested: omega-agatoxin IVA (200 nM) blocked only 13% of the Ca channel current, while omega-conotoxin GVIA (1 microM) blocked 84% of the current. Immnohistochemistry supported the presence of alpha1A, alpha1B, alpha1C, and alpha1D Ca channel subunits. Mapping of dihydropyridine-binding sites with DM-BODIPY revealed a distribution of channels over the entire membrane of the Muller cell with a higher density at the apical region. Overall, these observations suggest either the presence of a mix of L- and N-type Ca channels or a single, unconventional HVA Ca channel subtype sharing L- and N-type Ca channel characteristics.

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation.

It has been shown that β auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of β3 subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed β3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at “physiological” holding potentials (HPs) of −60 and −80 mV. At a HP of −80 mV, coinjection of various concentrations (0–12.5 ng) of the β3 with Cav2.2α1 and α2δ enhanced the maximum conductance of expressed channels at lower β3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The β3-induced current suppression was reversed at a HP of −120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of β3 at −80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and β3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel β3 effect. Steady-state inactivation curves revealed that N-type channels exhibited “closed-state” inactivation without β3, and that β3 caused an ∼40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately −85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of β3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at −80 mV. Similar “ultra-slow” inactivation was observed for N-type channels without β3. Thus, β3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting “ultra-slow” and “closed-state” inactivation properties.

Inhibition of the activity of T lymphocyte Kv1.3 channels by extracellular zinc

The inhibitory effect of zinc on voltage-gated Kv1.3 channels in human T lymphocytes was investigated using the “whole-cell” patch-clamp technique. Application of 10 and 20 μM Zn caused a concentration-dependent shift of activation midpoint of the whole-cell currents from −19.65±1.03 mV (mean±SE) under control conditions to 9.84±0.66 mV upon application of 20 μM Zn. This effect was saturated at zinc concentrations higher than 20 μM. The activation rate was considerably slower, whereas the deactivation rate was not significantly affected by Zn. Inactivation midpoint was shifted from −53.06±0.44 mV under control conditions to −36.05±0.48 mV in the presence of 100 μM Zn. Inactivation rate was not significantly affected upon Zn treatment. Whole-cell potassium currents were reduced to about 70% of their control values with no clear concentration dependence in the zinc concentration range from 10 to 100 μM. When raising the zinc concentration to levels above 100 μM, a concentration-dependent inhibition of the whole-cell currents appeared additionally to the changes in channel gating. The channels were half-blocked at the zinc concentration of 346±40 μM and the Hill slope coefficient was 1.89±0.21. The inhibitory effect of zinc was not complete at micromolar concentrations and was saturated at concentrations higher than 1 mM. This inhibitory effect was not accompanied by any further modification in the shift of the activation and inactivation midpoints nor by a slowing of the channel activation rate. The inhibitory effect of zinc was significantly diminished in the presence of 150 mM K + in the extracellular solution, whereas the zinc-induced shift of the activation threshold and slowing of the activation kinetics remained unchanged when raising extracellular potassium concentration. It is suggested that zinc acts on two independent binding sites on the channels. Binding to one site that is saturated at concentrations higher than 20 μM affects the channel gating. Binding to another site at concentrations higher than 100 μM inhibits the currents without affecting the channel gating.

Guanidine hydrochloride-induced reversible unfolding of sheep liver serine hydroxymethyltransferase

Equilibrium unfolding studies of sheep liver tetrameric serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) revealed that the enzyme assumed apparent random coil structure above 3 M guanidine hydrochloride (GdnHCl). In the presence of non-ionic detergent Brij-35 and polyethylene glycol, the 6 M GdnHCI unfolded enzyme could be completely (> 95%) refolded by a 40-fold dilution. The refolded enzyme was fully active and had kinetic constants similar to the native enzyme. The midpoint of inactivation (0.12 M GdnHCl) was well below the midpoint of unfolding (1.6±0.1 M GdnHCl) as monitored by far UV CD at 222 nm. In the presence of PLP, the midpoint of inactivation shifted to a higher concentration of GdnHCl (0.6 M) showing that PLP stabilizes the quaternary structure of the enzyme. However, 50% release of pyridoxal-5′-phosphate (PLP) from the active site occurred at a concentration (0.6 M) higher than the midpoint of inactivation suggesting that GdnHCl may also act as a competitive inhibitor of the enzyme at low concentrations which was confirmed by activity measurements. PLP was not required for the initiation of refolding and inactive tetramers were the end products of refolding which could be converted to active tetramers upon the addition of PLP. Size exclusion chromatography of the apoenzyme showed that the tetramer unfolds via the intermediate formation of dimers. Low concentrations (0.3–0.6 M) of GdnHCl stabilized at least one intermediate which was in slow equilibrium with the dimer. The binding of ANS was maximum at 0.4–0.6 M GdnHCl suggesting that the unfolding intermediate that accumulates at this concentration is less compact than the native enzyme.

Biophysical Characteristics of Swelling-Activated Cl − Channels in Human Tracheal 9HTEo-Cells

The question of whether a single molecule can account for every observed swelling-activated Cl- current deserves to be addressed and biophysical description seems to be an adequate criterion to classify these channels. We studied the biophysical properties of swelling-activated Cl- currents in 9HTEo-cells using whole-cell and outside-out patch clamp recordings. Hypotonic shock activated outwardly rectifying currents that inactivated at potentials higher than 20 mV. The decay phase of the current was well fitted by two exponential functions and both time constants were voltage-dependent. Two voltage-dependent time constants were also necessary to describe reactivation. The midpoint of current inactivation was 54 mV. The voltage dependence of kinetics did not significantly change by modifying the extracellular NaCl concentration while the inactivation midpoint slightly shifted. In conclusion, our results indicate that the voltage-dependent properties of the swelling-activated Cl- currents in 9HTEo- cells are largely independent from the extracellular ionic strength and the extracellular Cl- concentration. Excised patches from cells exposed to hypotonic shock showed single channel currents that inactivated at positive membrane potentials and displayed chord conductance of approximately 60 pS at 100 mV and of approximately 20 pS at -80 mV. The permeability sequence for the single channel was I- > Br- > Cl- > gluconate and currents were blocked by Reactive blue 2. These properties indicate that intermediate conductance outwardly rectifying channels are responsible for the macroscopic swelling-activated current.

Reversible unfolding of sheep liver tetrameric serine hydroxymethyltransferase

Equilibrium unfolding studies of the tetrameric serine hydroxymethyltransferase from sheep liver (SHMT, E.C.2.1.2.1) revealed that the holoenzyme, apoenzyme and the sodium borohydride-reduced holoenzyme had random coil structures in 8 M urea. In the presence of a non-ionic detergent, Brij-35, and polyethylene glycol, the 8 M urea unfolded protein could be completely (>95%) refolded by a 20-fold dilution. The refolded enzyme was completely active and kinetically similar to the native enzyme. The midpoint of inactivation of the enzyme occurred at a urea concentration that was much below the urea concentration required to bring about a substantial loss of secondary structure. This observation suggested the occurrence of a `predenaturation transition' in the unfolding pathway. The equilibrium urea-induced denaturation curve of holoSHMT showed two transitions. The midpoint of the first transition was 1.2 M, which was comparable to that required for 50% decrease in enzyme activity. Further, 50% release of the pyridoxal-5′-phosphate (PLP) from the active site, as monitored by decrease in absorbance at 425 nm, also occurred at about 1.2 M urea. Size exclusion chromatography showed that the tetrameric SHMT unfolds via the intermediate formation of dimers. This dissociation occurred at a much lower urea concentration (0.15 M) in the unfolding of the apoenzyme, and at a higher urea concentration (1.2 M) in the unfolding of holoenzyme, thereby demonstrating the involvement of PLP in stabilizing the quaternary structure of the enzyme. Size exclusion chromatography of the refolding intermediates demonstrated that the cofactor shifts the equilibrium towards the formation of the active tetramer. The reduced holoenzyme could also be refolded to its native structure, as observed by fluorescence and CD measurements, indicating that the presence of covalently linked PLP does not affect refolding. The results demonstrate clearly that the dimer is an intermediate in the urea-induced equilibrium unfolding/refolding of sheep liver SHMT; and PLP, in addition to its role in catalysis, is required for the stabilization of the tetrameric structure of the enzyme.

CLINICAL CONCENTRATIONS OF ISOFLURANE SIGNIFICANTLY SUPPRESSES SODIUM CURRENTS IN RAT DORSAL ROOT GANGLIA

S519 Introduction: Over the last decade a number of studies have revealed that clinical concentrations of volatile anesthetics alter the discharge pattern and action potential properties of mammalian CNS and peripheral neurons [1]. In thin and unmyelinated neurons, which represent a substantial fraction of intracortical fibers and sensory afferents, anesthetics also have been found to alter signal propagation. [1] These neuronal functions all depend on the proper function of voltage-dependent sodium channels, making these integral membrane proteins potentially important molecular targets of general anesthetics. Although early studies examining anesthetic modification of sodium channel currents in non-mammalian preparations found them to be relatively insensitive targets compared with ligand-gated channels, a recent report has found that rat brain-Ila sodium channels expressed in a mammalian cell line are as sensitive as GABA-A receptors to clinical concentrations of volatile anesthetics [2]. It therefore has become essential to determine whether sodium channels in mammalian neurons have a similar anesthetic sensitivity as the expressed channels. To examine this question, we examined isoflurane modification of sodium currents in cultured rat Dorsal Root Ganglia (DRG). Methods: Freshly dissociated and primary cultured neurons of DRG from 5-30 day rats were obtained after enzymatic digestion with collagenase and/or trypsin. Sodium currents were measured using the whole cell patch clamp configuration; the extracellular Hanks' balanced salt solution additionally contained 5 mM HEPES and 0.1 mM CdCl2, pH 7.2, while the internal solution was 140 mM CsF, 10 mM NaCl, 5 mM HEPES, pH 7.4. Control measurements of the cells elicited normal action potentials and ionic currents. Sodium currents and anesthetic suppression were measured with depolarizing test potentials from a hyperpolarizing holding potential. Aqueous concentrations of isoflurane, which are relatively independent of experimental temperature [3], were measured by gas chromatography. All experiments were conducted at room temperature (22-25[degree sign]C). Results: Similar to results with the rat brain Ila sodium channels, isoflurane suppressed sodium currents through at least two mechanisms: a) a voltage-independent suppression of resting or open channels at hyperpolarized potentials, and b) a hyperpolarizing shift in the voltage-dependence of channel inactivation. Unlike the expressed channels, however, even high concentrations of isoflurane could not completely block sodium currents at potentials more negative than -80 mV. At -80 mV, isoflurane maximally suppressed 54 +/- 7.0% of sodium current (n=11, concentrations up to 5.4 mM isoflurane). The range of maximal suppression was 40-75%. Averaging data from the 11 cells and fitting to a rectangular hyperbola yielded a maximal suppression of 0.49 with an ED50=0.17 mM, slightly lower than aqueous MAC concentrations. The midpoint of sodium channel inactivation was about -50 mV. Similar to rat brain sodium channels, 0.54 mM isoflurane (twice MAC) shifted the inactivation midpoint by about -20 mV, resulting in significantly greater current suppression at these potentials (about 80% block at -60mV, compared with about 20% at -80 mV). Conclusions: Clinical concentrations of isoflurane caused a large depolarizing shift in steady-state channel inactivation, resulting in the block of a significant fraction (>50%) of sodium currents in DRG neurons at resting membrane potentials. These results support the hypothesis that some mammalian neuronal sodium channels are sensitive to clinical concentrations of volatile anesthetics in situ, and that their anesthetic modification may directly alter neuronal function.

Enhanced Closed-state Inactivation in a Mutant Shaker K + Channel

Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed "multiple-state inactivation" model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels.