Half Inactivation Research Articles

Molecular and Functional Changes in Voltage-Gated Na+ Channels in Cardiomyocytes During Mouse Embryogenesis

Embryonic cardiomyocytes undergo profound changes in their electrophysiological properties during development. However, the molecular and functional changes in Na⁺ channel during cardiogenesis are not yet fully explained. To study the functional changes in the Na⁺ channel during cardiogenesis, Na⁺ currents were recorded in the early (EDS) and late (LDS) developmental stages of cardiomyocytes in embryonic mice. Compared with EDS myocytes, LDS myocytes exhibited a larger peak current density, a more negative shift in the voltage of half inactivation, a larger fast inactivation component and a smaller slow inactivation component, and smaller time constants for recovery from inactivation. Additionally, multiple Na⁺ channel α-subunits (Nav 1.1-1.6) and β-subunits (Nav β1-β3) of mouse embryos were investigated. Transcripts of Nav 1.1-1.3 were absent or present at very low levels in embryonic hearts. Transcripts encoding Nav 1.4-1.6 and Nav β1-β3 increased during embryogenesis. Data on the sensitivity of total Na⁺ currents to tetrodotoxin (TTX) showed that TTX-resistant Nav 1.5 is the predominant isoform expressed in the heart of the mouse embryo. The results indicate that significant changes in the functional properties of Na⁺ channels develop in the cardiomyocytes of the mouse embryo, and that different Na⁺ channel subunit genes are strongly regulated during embryogenesis, which further support a physiological role for voltage-gated Na⁺ channels during heart development.

Targeting atrioventricular differences in ion channel properties for terminating acute atrial fibrillation in pigs

The goal was to terminate atrial fibrillation (AF) by targeting atrioventricular differences in ionic properties. Optical mapping was used to record electrical activity during carbachol (0.25-0.5 μM)-induced AF in pig hearts. The atrial-specific current, I(Kur), was blocked with 100 μM 4-aminopyridine (4-AP) or with 0.5 μM DPO-1. Hearts in AF and ventricular fibrillation (VF) were also subjected to increasing levels of extracellular K(+) ([K(+)](o): 6-12 mM), compared with controls (4 mM). We hypothesized that due to the more negative steady-state half inactivation voltage for the atrial Na(+) current, I(Na), compared with the ventricle, AF would terminate before VF in hyperkalaemia. Mathematical models were used to interpret experimental findings. The I(Kur) block did not terminate AF in a majority of experiments (6/9 with 4-APand 3/4 with DPO-1). AF terminated in mild hyperkalaemia ([K(+)](o) ≤ 10.0 mM; N = 8). In contrast, only two of five VF episodes terminated at the maximum ([K(+)](o): 12 mM [K(+)](o)). The I(Kur) block did not terminate a simulated rotor in cholinergic AF because its contribution to repolarization was dwarfed by the large magnitude of the acetylcholine-activated K(+) current (I(K,ACh)). Simulations showed that the lower availability of the atrial Na(+) current at depolarized potentials, and a smaller atrial tissue size compared with the ventricle, could partly explain the earlier termination of AF compared with VF during hyperkalaemia. I(Kur) is an ineffective anti-arrhythmic drug target in cholinergic AF. Manipulating Na(+) current 'availability' might represent a viable anti-arrhythmic strategy in AF.

Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai

Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28 and 33kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40°C, respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was also similar to each other, i.e., 38°C. However, NaCl requirement and activity toward oligosaccharide substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than 0.2 M, whereas AkAly33 showed ~20% of maximal activity despite the absence of NaCl and the maximal activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide, while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes belong to polysaccharide lyase family 14.

Purification and kinetic characterization of the liverwort Pallavicinia lyelli (Hook.) S. Gray. cytosolic ascorbate peroxidase

Ascorbate peroxidase (APX) of the liverwort Pallavicinia lyelli was extracted and purified through ammonium sulfate precipitation, Butyl-Toyopearl, DEAE-Cellulofine and Sephadex G-75 chromatography. The purification factor for APX was 285 with 7.9% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of APX was approximately 28 kDa estimated by SDS-PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 6.0. The enzyme had a temperature optimum at 40 degrees C and was relatively stable at 60 degrees C, with 54% loss of activity. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity and the capacity to utilize alternate electron donors. p-Chloromercuribenzoate (pCMB), hydroxyurea and salicylic acid (SA) significantly inhibited APX activity. Ascorbate (AsA) and pyrogallol were found to be efficient substrates for Pallavicinia APX, considering the Vmax/Km ratio. We detected the activity of monodehydroascorbate reductase (MDHAR) involved in the regeneration of ascorbate, but failed to detect the dehydroascorbate reductase (DHAR) activity. The data obtained in this study may help to understand desiccation tolerance mechanism in the liverwort.

Systematic comparison of intrinsic membrane properties of locus coeruleus neurons in Mecp2‐null and wild‐type mice

Rett syndrome caused by mutations in MECP2 (Methyl‐CpG binding protein 2) gene shows autonomic abnormalities, in which brainstem norepinephrinergic (NE) systems are involved. To determine if the Mecp2 defect affects intrinsic membrane properties of locus coeruleus (LC) NE neurons, whole‐cell current clamp was performed in brain slices of 3‐4 weeks old Mecp2‐/y and wild‐type mice. Of 102 neurons studied, no statistical differences were found in passive membrane properties except the time constant that was significantly smaller in Mecp2‐/y neurons. Most LC neurons in both groups displayed spontaneous firing activity. The action potential duration was prolonged in Mecp2‐/y mice due to an extended rising phase. Voltage clamp in acutely dissociated LC neurons confirmed that the density of voltage‐activated Na+ currents was reduced in Mecp2‐/y mice. Mecp2 knockout produced a negative shift of half inactivation potential of delayed excitation, and had rather small effect on spike frequency adaption. The overall amplitude of afterhyperpolarization (AHP) was lower in Mecp2‐/y neurons with a clear manifestation of the fast AHP. No evident bursting activity and post‐inhibition rebound was observed in both groups. The electrophysiological abnormalities of LC NE neurons involving likely defects in Na+ and K+ channels seem to contribute to the autonomic dysfunction in Mecp2‐/y mice. (supported by NIH R01‐HL067890)

Cyclophosphamide‐evoked heart failure involves pronounced co‐suppression of cytoplasmic thioredoxin reductase activity and non‐protein free thiol level

Heart failure is a life-threatening complication of high-dose cyclophosphamide (CTX) chemotherapy, and the present study aimed at identifying the mechanism involved in mice. CTX at 800 mg/kg resulted in heart failure, in which cytoplasmic thioredoxin reductase (TrxR1) activity and non-protein free thiol (NPFT) level were suppressed by 90 and 62%, respectively. The combination of 350 mg/kg CTX and the glutathione synthesis inhibitor buthionine sulfoximine (BSO) also evoked heart failure, in which TrxR1 activity and NPFT level were suppressed by 66 and 62%, respectively. NPFT depletion alone by BSO did not cause cardiac toxicity. CTX at 350 mg/kg alone also did not cause cardiac toxicity, even though it suppressed TrxR1 activity by 68%. Previous studies have shown that half inactivation of TrxR1 in tumour, bladder, and kidneys was associated with toxicological consequences. Cardiac TrxR1 is dispensable, but cardiac cytoplasmic thioredoxin (Trx1) is essential. The potential uncoupling between TrxR1 and Trx1 may explain why there is no cardiac toxicity following TrxR1 inhibition. However, TrxR1 inactivation may still play a role in CTX-evoked heart failure because inactivated TrxR1 gains cytotoxic function, which may engender noticeable toxicity when massive NPFT is deleted. CTX-evoked heart failure involves pronounced co-suppression of TrxR1 activity and NPFT level.

Differential cold–adaptation among protein components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125

Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

Sodium current kinetics of transitional myocytes in Koch triangle of rabbit hearts

Few studies have explored the inward sodium current (INa) kinetics of transitional cardiomyocytes. This study aimed to explore the kinetics of transitional cardiomyocytes types alpha and beta. The whole-cell patch clamp technique was used to study the rapid INa of isolated transitional cardiomyocytes in the Koch triangle of rabbit hearts. Maximal amplitude and density of INa in type alpha and type beta was (-1627 +/- 288) pA (alpha), (-35.17 +/- 6.56) pA/pF (beta) and (-3845 +/- 467) pA (alpha), (-65.64 +/- 10.23) pA/pF (beta) (P < 0.05). Steady state activation curves of INa, fitted to a Boltzmann distribution for both types, were sigmoid in shape. Half activation voltage and slope factors did not significantly differ between types at (-43.46 +/- 0.85) mV (alpha), (-41.39 +/- 0.47) mV (beta) or (9.04 +/- 0.66) mV (alpha), (11.08 +/- 0.89) mV (beta). Steady state inactivation curves of INa, fitted to a Boltzmann distribution in both types were inverse "S" shape. Half inactivation voltage and slope factors were (-109.9 +/- 0.62) mV (alpha), (-107.5 +/- 0.49) mV (beta) and (11.78 +/- 0.36) mV (alpha), (11.57 +/- 0.27) mV(beta), (P > 0.05), but time constants of inactivation were significantly different at (1.10 +/- 0.19) mV (alpha) and (2.37 +/- 0.33) ms (beta), (P < 0.05). Time constants of recovery from inactivation of INa for both types were (122.16 +/- 27.43) mV (alpha) and (103.84 +/- 28.97) ms (beta) (P < 0.05). Transitional cardiomyocytes in rabbit hearts show a heterogeneous, voltage gated and time dependent fast inward sodium current. Types alpha and beta show the features of INa similar to those in slow- and fast-response myocytes, with probably better automaticity and conductivity, respectively.

Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition

Transformation with the bacterial gene codA for choline oxidase allows Synechococcus sp. PCC 7942 cells to accumulate glycinebetaine when choline is supplemented exogenously. First, we observed two types of protective effect of glycinebetaine against heat-induced inactivation of photosystem II (PSII) in darkness; the codA transgene shifted the temperature range of inactivation of the oxygen-evolving complex from 40–52 °C (with half inactivation at 46 °C) to 46–60 °C (with half inactivation at 54 °C) and that of the photochemical reaction center from 44–55 °C (with half inactivation at 51 °C) to 52–63 °C (with half inactivation at 58 °C). However, in light, PSII was more sensitive to heat stress; when moderate heat stress, such as 40 °C, was combined with light stress, PSII was rapidly inactivated, although these stresses, when applied separately, did not inactivate either the oxygen-evolving complex or the photochemical reaction center. Further our studies demonstrated that the moderate heat stress inhibited the repair of PSII during photoinhibition at the site of synthesis de novo of the D1 protein but did not accelerate the photodamage directly. The codA transgene and, thus, the accumulation of glycinebetaine alleviated such an inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein. We propose a hypothetical scheme for the cyanobacterial photosynthesis that moderate heat stress inhibits the translation machinery and glycinebetaine protects it against the heat-induced inactivation.

Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W

Congenital stationary night blindess-2 (incomplete congenital stationary night blindness (iCSNB) or CSNB-2) is a nonprogressive, X-linked retinal disease which can lead to clinical symptoms such as myopia, hyperopia, nystagmus, strabismus, decreased visual acuity, and impaired scotopic vision. These clinical manifestations are linked to mutations found in the CACNA1F gene which encodes for the Ca v1.4 voltage-gated calcium channel. To better understand the physiological effects of these mutations, three missense mutants, F742C, G1007R and R1049W, previously shown to be mutated in patients with CSNB-2, were transiently expressed in human embryonic kidney (HEK) tsA-201 cells and characterized using whole-cell patch clamp. The G1007R mutation is located in transmembrane segment 5 (S5) of domain III and R1049W is located in the extracellular linker between S5 and the P-loop of domain III. Both mutants produced full length proteins that targeted to the membrane but did not support ionic currents. In 20 mM Ba 2+, F742C (S6 domain II) produced a ∼21 mV hyperpolarizing shift in half activation potential (V a[1/2]) and a ∼23 mV hyperpolarizing shift in half inactivation potential (V h[1/2]). Additionally, F742C displayed slower inactivation kinetics and a smaller whole cell conductance (G max). In physiological 2 mM Ca 2+, F742C produced a ∼19 mV hyperpolarizing shift in V a[1/2]. These findings suggest that the pathology of CSNB-2 in patients with these missense mutations in the Ca v1.4 calcium channel is the result in either a gain of function (F742C) or a loss of function (G1007R, R1049W).

Molecular and functional properties of the psychrophilic elongation factor G from the Antarctic Eubacterium Pseudoalteromonas haloplanktis TAC 125

The molecular and functional properties of the elongation factor (EF) G from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) were studied. PhEF-G catalyzed protein synthesis in vitro that was inhibited by fusidic acid, an antibiotic specifically acting on EF-G. The EF interacted with GDP only in the presence of P. haloplanktis ribosome and fusidic acid with an affinity similar to that displayed by Escherichia coli EF-G. The psychrophilic translocase elicited a ribosome-dependent GTPase that was competitively inhibited by GDP, the slowly hydrolyzable GTP analog GppNHp, and the protein synthesis inhibitor ppGDP. The temperature dependence of the activity of PhEF-G reached its maximum at least 26 degrees C beyond the growth temperature of P. haloplanktis (4-20 degrees C). The heat inactivation profile of the ribosome-dependent GTPase of PhEF-G gave a temperature for half inactivation (46 degrees C), significantly lower than that for half denaturation measured by either UV- (57 degrees C) or fluorescence-melting (62 degrees C). This finding was attributed to a different effect of the temperature on the catalytic domain with respect to that elicited on the other domains constituting the EF, thus confirming the differential molecular flexibility present in psychrophilic enzymes. A molecular model, based on the 3D coordinates of a thermophilic EF-G, showed differences only in connecting loops.

Myocytes of resistance‐size arteries express Ca V 1.2 channels with a novel N‐terminus

Voltage-dependent L-type Ca2+ channels (CaV1.2) are the principal Ca2+ entry pathway in myocytes of small arteries that regulate vascular pressure and blood flow. However, the molecular identity of the pore-forming a1 subunit (CaV1.2) expressed in myocytes of myogenic arteries is unclear. Here, we cloned CaV1.2 subunits from rat resistance-sized cerebral arteries and found they contain a novel, cysteine rich N-terminus that derives from exon 1 (termed exon 1c), which is located within CACNA1C, the CaV1.2 gene. Another 5′ end sequence (exon 1b) identical to the brain CaV1.2 5′ end was also detected. Exon 1c was predominant in arterial myocytes, but barely present in cardiac myocytes where exon 1a prevailed. When co-expressed with a2d subunits, macroscopic currents generated from channels containing the exon 1c-derived N-terminus exhibited a smaller IBa density and more negative voltages of half activation (V1/2,act) and half inactivation (V1/2,inact) when compared with those containing exon 1b. Co-expression of ß subunits increased IBa density, caused hyperpolarizing shifts in V1/2,act and V1/2,inact, and eliminated differences observed when channels were expressed with only a2d. Our data indicate that modulation of CaV1.2 currents mediated by a novel CaV1.2 N-terminus may lead to tissue specific Ca2+ signaling in myocytes of resistance-size arteries.

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg 2+ concentration on L-type Ca 2+ ( I Ca) and Ba 2+ currents ( I Ba) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8 mM [Mg 2+] ([Mg 2+] p) buffered with 30 mM citrate and 10 mM ATP. Increasing [Mg 2+] p from 0.2 to 1.8 mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current–voltage relationships were studied with two-pulse and tail current ( I T) protocols, respectively. Increasing [Mg 2+] p shifted the V M for half inactivation by −20 mV but dramatically decreased I Ca amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I T amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg 2+] p might also inhibit permeation through open Ca 2+ channels at positive V M.

Modulation of A-type potassium currents in retinal horizontal cells by extracellular calcium and zinc

Extracellular Ca2+ and Zn2+ influence many aspects of retinal function. Here, we examined the effect of external Ca2+ and Zn2+ on potassium channels of retinal horizontal cells. When extracellular Ca2+ was lowered from 3 mM to 0.3 mM, horizontal cell transient outward currents elicited by voltage steps from resting membrane potential (-70 mV) were decreased by approximately 50%, whereas the sustained currents remained unchanged. This effect was due to a hyperpolarizing shift in the steady-state inactivation curve of A-type K+ currents when extracellular Ca2+ concentration was lowered. The mean half inactivation potential of the steady-state inactivation curves was hyperpolarized from -56.3 +/- 4.7 mV in 3 mM Ca2+ to -76.4 +/- 3.9 mV in 0.3 mM Ca2+. Neither the state-steady activation curve nor the kinetics of inactivation was significantly changed in low extracellular Ca2+. The addition of 30 microM Zn2+ restored peak outward currents in 0.3 mM Ca2+. The half inactivation voltages were depolarized from -70 +/- 2.8 mV in 0.3 mM Ca2+ to -56 +/- 2.6 mV in 0.3 mM Ca2+ plus 30 microM Zn2+. Taken together, the results indicate that external Ca2+ and Zn2+ maintain the activity of A-type potassium channels in retinal horizontal cells by influencing the voltage dependence of steady-state inactivation.

Sulfur dioxide derivatives modulation of high-threshold calcium currents in rat dorsal root ganglion neurons

This study addressed the effect of sulfur dioxide (SO 2) derivatives on high-voltage-activated calcium currents (HVA- I Ca) in somatic membrane of freshly isolated rat dorsal root ganglion (DRG) neurons by using the whole-cell configuration of patch-clamp technique. High-threshold Ca 2+ channels are highly expressed in small dorsal root ganglion neurons. SO 2 derivatives increased the amplitudes of calcium currents in a concentration-dependent and voltage-dependent manner. The 50% enhancement concentrations (EC 50) of SO 2 derivatives on HVA- I Ca was about 0.4 μM. In addition, SO 2 derivatives significantly shifted the activation and inactivation curve in the depolarizing direction. Parameters for the fit of a Boltzmann equation to mean values for the activation were V 1/2 = −17.9 ± 1.3 mV before and −12.5 ± 1.1 mV after application 0.5 μM SO 2 derivatives 2 min ( P < 0.05). The half inactivation of HVA- I Ca was shifted 9.7 mV to positive direction ( P < 0.05). Furthermore, SO 2 derivatives significantly prolonged the slow constant of inactivation, slowed the fast recovery but markedly accelerated the slow recovery of HVA- I Ca from inactivation. From HP of −60 mV 0.5 μM SO 2 derivatives increased the amplitude of HVA- I Ca with a depolarizing voltage step to −10 mV about 54.0% in small DRG neurons but 33.3% in large DRG neurons. These results indicated a possible correlation between the change of calcium channels and SO 2 inhalation toxicity, which might cause periphery neurons abnormal regulation of nociceptive transmission via calcium channels.

Opioid μ receptor activation inhibits sodium currents in prefrontal cortical neurons via a protein kinase A- and C-dependent mechanism

Opioid transmission in the medial prefrontal cortex is involved in mood regulation and is altered by drug dependency. However, the mechanism by which ionic channels in cortical neurons are controlled by μ opioid receptors has not been elucidated. In this study, the effect of μ opioid receptor activation on voltage-dependent Na + currents was assessed in medial prefrontal cortical neurons. In 66 out of 98 nonpyramidal neurons, the application of 1 μM of DAMGO ([ d-Ala 2, N-Me-Phe 4, Gly 5-OL]-enkephalin), a specific μ receptor agonist, caused a decrease in the Na + current amplitude to approximately 79% of that observed in controls (half blocking concentration = 0.094 μM). Moreover, DAMGO decreased the maximum current activation rate, prolonged its time-dependent inactivation, and shifted the half inactivation voltage from −63.4 mV to −71.5 mV. DAMGO prolonged the time constant of recovery from inactivation from 5.4 ms to 7.4 ms. The DAMGO-evoked inhibition of Na + current was attenuated when GDP-β-S (0.4 mM, Guanosine 5-[β-thio]diphosphate trilithium salt) was included in the intracellular solution. Inhibitors of kinase A and C greatly attenuated the DAMGO-induced inhibition, while adenylyl cyclase and kinase C activators mirrored the DAMGO inhibitory effect. Na + currents in pyramidal neurons were insensitive to DAMGO. We conclude that the activation of μ opioid receptors inhibits the voltage-dependent Na + currents expressed in nonpyramidal neurons of the medial prefrontal cortex, and that kinases A and C are involved in this inhibitory pathway.

Effects of ATP and GTP on voltage-gated K + currents in glandular and muscular sympathetic neurons

This study assesses the effects of ATP and GTP on the kinetic properties of voltage-gated K + currents in anatomically identified postganglionic sympathetic neurons innervating the submandibular gland and the masseter muscle in rats. Three types of K + currents were isolated: the I Af steady-state inactivating at more hyperpolarized potentials, I As steady-state inactivating at less hyperpolarized potentials than I Af and the I K current independent of membrane potential. The kinetic properties of these currents were tested in neurons with ATP (4 mM) and GTP (0.5 mM) or without ATP and GTP in the intracellular solution.In glandular and muscular neurons in the absence of ATP and GTP in the intracellular solution, the current density of I Af was significantly larger (142 pA/pF and 166 pA/pF, respectively) comparing to cells with ATP and GTP (96 pA/pF and 100 pA/pF, respectively). The I As was larger only in glandular neurons (52 pA/pF vs. 37 pA/pF).Conversely, I K current density was smaller in glandular and muscular neurons without ATP and GTP (17 pA/pF and 31 pA/pF, respectively) comparing to cells with ATP and GTP (57 pA/pF and 58 pA/pF, respectively). In glandular (15.5 nA/ms vs. 6.9 nA/ms) and muscular (10.9 nA/ms vs. 7.5 nA/ms) neurons, the I Af activated faster in the absence of ATP and GTP. Half inactivation voltage of I Af in glandular (−110.0 mV vs. −119.7 mV) and muscular (−108.4 vs. −117.3 mV) neurons was shifted towards depolarization in the absence of ATP and GTP. We suggest that the kinetic properties of K + currents in glandular and muscular sympathetic neurons change markedly in the absence of ATP and GTP in the cytoplasm. Effectiveness of steady-state inactivated currents ( I Af and I AS) increased, while effectiveness of steady-state noninactivated currents decreased in the absence of ATP and GTP. The effects were more pronounced in glandular than in muscular neurons.

Sodium and calcium currents shape action potentials in immature mouse inner hair cells.

Before the onset of hearing at postnatal day 12, mouse inner hair cells (IHCs) produce spontaneous and evoked action potentials. These spikes are likely to induce neurotransmitter release onto auditory nerve fibres. Since immature IHCs express both alpha1D (Cav1.3) Ca2+ and Na+ currents that activate near the resting potential, we examined whether these two conductances are involved in shaping the action potentials. Both had extremely rapid activation kinetics, followed by fast and complete voltage-dependent inactivation for the Na+ current, and slower, partially Ca2+-dependent inactivation for the Ca2+ current. Only the Ca2+ current is necessary for spontaneous and induced action potentials, and 29 % of cells lacked a Na+ current. The Na+ current does, however, shorten the time to reach the action-potential threshold, whereas the Ca2+ current is mainly involved, together with the K+ currents, in determining the speed and size of the spikes. Both currents increased in size up to the end of the first postnatal week. After this, the Ca2+ current reduced to about 30 % of its maximum size and persisted in mature IHCs. The Na+ current was downregulated around the onset of hearing, when the spiking is also known to disappear. Although the Na+ current was observed as early as embryonic day 16.5, its role in action-potential generation was only evident from just after birth, when the resting membrane potential became sufficiently negative to remove a sizeable fraction of the inactivation (half inactivation was at -71 mV). The size of both currents was positively correlated with the developmental change in action-potential frequency.

APRT from erythrocytes of HGPRT deficient patients: kinetic, regulatory and thermostability properties.

Adenine phosphoribosyltransferase (APRT) has been 1200-fold purified from erythrocytes of a patient with partial hipoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, Propositus, and in those of a controlHPRT+, with 20% efficiency in both proteins and specific activity of 550 and 243 nmol/h/mgprotein. The specific activity determined in the Propositus enzyme was, in all purification steps, higher than that of the controlHPRT+. Significant changes were found in their thermal stabilities. Half inactivation times at each temperature studied are greater for the Propositus enzyme in the temperature interval 60-80 degrees C. No significant difference has been observed in the affinity constants for adenine and PRPP substrates. Studies on inhibition by the reaction product suggest that AMP is a competitive inhibitor with respect to PRPP in both enzymes, with Ki values of 150 microM in Propositus and 220 microM in controlHPRT+.

KChIP1 and frequenin modify shal-evoked potassium currents in pyloric neurons in the lobster stomatogastric ganglion.

The transient potassium current (I(A)) plays an important role in shaping the firing properties of pyloric neurons in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. The shal gene encodes I(A) in pyloric neurons. However, when we over-expressed the lobster Shal protein by shal RNA injection into the pyloric dilator (PD) neuron, the increased I(A) had somewhat different properties from the endogenous I(A). The recently cloned K-channel interacting proteins (KChIPs) can modify vertebrate Kv4 channels in cloned cell lines. When we co-expressed hKChIP1 with lobster shal in Xenopus oocytes or lobster PD neurons, they produced A-currents resembling the endogenous I(A) in PD neurons; compared with currents evoked by shal alone, their voltage for half inactivation was depolarized, their kinetics of inactivation were slowed, and their recovery from inactivation was accelerated. We also co-expressed shal in PD neurons with lobster frequenin, which encodes a protein belonging to the same EF-hand family of Ca(2+) sensing proteins as hKChIP. Frequenin also restored most of properties of the shal-evoked currents to those of the endogenous A-currents, but the time course of recovery from inactivation was not corrected. These results suggest that lobster shal proteins normally interact with proteins in the KChIP/frequenin family to produce the transient potassium current in pyloric neurons.