Thiamine phosphorylated derivatives and bioelectrogenesis

Kinetic as well as thermodynamic considerations favour the idea that the change in sodium conductance explaining the action potential, must result from a bimolecular reaction system. The fact that thiamine phosphorylated derivatives are associated with the specific protein forming the sodium channel could well mean that these thiamine derivatives and more specifically thiamine triphosphate are directly involved in the conductance change.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

The interactive effects of fatigue and pH on the ionic conductance of frog sartorius muscle fibers

The effects of fatigue on the membrane conductance of frog sartorius muscle at the resting potential and during an action potential were studied. When muscles were exposed to an extracellular pH of 8.0 the membrane conductance at the resting potential increased during fatigue by about 20% and returned to prefatigue level in about 20 min. The membrane conductance of muscle fibers exposed to pH 6.4 was about three times less than that of pH 8.0 and decreased further during fatigue. Furthermore, the recovery of a normal membrane conductance was slow at pH 6.4. Both the inward, depolarizing and the outward, repolarizing currents during the action potential are reduced in fatigue. In each case the effect is greater at pH 6.4 than at 8.0 and recovery towards normal values is slower at pH 6.4. It is concluded that the ionic conductance of the sarcolemmal membrane at the resting potential and during an action potential are modified by fatigue and that these changes are modulated by pHo.     

Low-temperature induced transmembrane potential changes in the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that the liverwort Conocephalum conicum generates all-or-none action potentials (APs) in response to a sudden temperature drop. In plants with anion and potassium conductance blocked, dose-dependent voltage transients (VTs) were evoked by cold stimuli. These VTs did not propagate. When the external concentration of Ca(2+) was decreased or calcium channel inhibitors (La(3+), Gd(3+), verapamil, Mg(2+), Mn(2+)) were used, inhibition of VTs was observed. Amplitudes of both APs and VTs grew when Sr(2+) ions, known to release calcium from internal stores, were added to the medium. Neomycin, which suppresses phospholipase C and indirectly affects inositol triphosphate formation, caused substantial inhibition of both APs and VTs. It is concluded that a temperature drop elucidated membrane potential changes due to calcium influx both from external and internal stores.     

Ionic basis for excitability of normal rat kidney (NRK) fibroblasts

Ionic membrane conductances of normal rat kidney (NRK) fibroblasts were characterized by whole-cell voltage-clamp experiments on single cells and small cell clusters and their role in action potential firing in these cells and in monolayers was studied in current-clamp experiments. Activation of an L-type calcium conductance (GCaL) is responsible for the initiation of an action potential, a calcium-activated chloride conductance (GCl(Ca)) determines the plateau phase of the action potential, and an inwardly rectifying potassium conductance (GKir) is important for the generation of a resting potential of approximately -70 mV and contributes to action potential depolarization and repolarization. The unique property of the excitability mechanism is that it not only includes voltage-activated conductances (GCaL, GKir) but that the intracellular calcium dynamics is also an essential part of it (via GCl(Ca)). Excitability was found to be an intrinsic property of a fraction (approximately 25%) of the individual cells, and not necessarily dependent on gap junctional coupling of the cells in a monolayer. Electrical coupling of a patched cell to neighbor cells in a small cluster improved the excitability because all small clusters were excitable. Furthermore, cells coupled in a confluent monolayer produced broader action potentials. Thus, electrical coupling in NRK cells does not merely serve passive conduction of stereotyped action potentials, but also seems to play a role in shaping the action potential.     

Cell response of Escherichia coli to cisplatin-induced stress

Cisplatin is undoubtedly one of the most common and successful anticancer drugs worldwide. Though its DNA-based mechanism of action is well established, the contribution of the proteome to this process remains unclear. The possible impact of particular Escherichia coli proteins on the cytostatic activity of cisplatin was the subject of this study. Our main focus was not only the "bottom-up" identification of novel cisplatin protein targets through LC/LC-MS/MS analysis, but also a label-free quantification of their regulation profile by spectral-counting. The regulation of two proteins, aconitate hydratase 2 and 60 kDa chaperonin 1, could be linked to a platinated amino acid in the protein sequence, whereas in the cases of 30S ribosomal protein S1 and enolase, it could be shown that cisplatin fragments are coordinated to an essential site for the functionality of the protein. Nucleoside triphosphate pyrophosphohydrolase (MazG) regulates the programmed cell death and was found to be platinated on the protein surface, which probably correlates with the established mode of action. A possible new chapter in the understanding of cisplatin's mechanism of action and its severe side effects is opened, since evidence is provided that platinated proteins are not only involved in cellular stress response but also in energy metabolism through glycolysis and catabolic processes, in gene regulatory mechanisms and protein synthesis.     

Spontaneous recovery of the anoxic diminution in the action potential plateau of the late embryonic and neonatal chicken hearts

The action potential plateau of the embryonic chick hearts at the latest stage of development (19- to 20-day-old) was depressed initially by anoxia but recovered spontaneously under the sustained anoxic condition. A similar spontaneous recovery of the action potential plateau was observed in hearts from neonatal chicks. Propranolol (3 × 10^?6M) did not affect the spontaneous recovery of the action potential plateau, excluding the possible involvement of the release of endogenous catecholamines. Since tissue adenosine triphosphate content continued to decrease during anoxia in these hearts, it is unlikely that the anoxia-resistant metabolic pathway appeared or was enhanced to supply the energy for the rebuilding of the plateau. In a Ca²⁺-free solution the action potential plateau rapidly disappeared and did not recover spontaneously during anoxia; at this time, however, the addition of Ca²⁺ (2 mM) prolonged the plateau. An inward calcium current may play an important role in spontaneous recovery of the action potential plateau during anoxia. The duration of the action potential decreased after hatching. From these results it is suggested that the mechanism underlying the action potential plateau may somehow differ in the hearts at the stages around the time of hatching.     

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue. Received: 5 May 1998 / Revised version: 27 October 1998 / Accepted: 19 January 1999     

Effects of morphine and meperidine on action potential production in frog's skeletal muscle fibers.

Morphine (3.3 times 10-minus 4 M) and meperidine (8.8 times 10-minus 5 M) inhibited action potential production in frog's skeletal muscle fibers. Over these concentration ranges, neither the resting membrane potentials nor the resting membrane electric properties of the fibers appeared to be modified. Both drugs depressed excitability and the rising phase of the action potential by inhibiting the specific increase in sodium conductance which normally follows an adequate stimulus. Both drugs also seemed to inhibit the secondary rise in potassium conductance which normally occurs during an action potential, causing a prolongation of the action potential duration.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

Action potential modulation of connexin40 gap junctional conductance

Connexin40 (Cx40) is abundantly expressed in the atrial myocardium, ventricular conduction system, and vascular endothelial and smooth muscle cells of the mammalian cardiovascular system. Rapid conduction through cardiac tissues depends on electrotonic transfer of the action potential between neighboring cells. To determine whether transjunctional voltages (Vj) elicited by an action potential can modulate conductance of Cx40 gap junctions, simulated myocardial action potentials were applied as voltage-clamp waveforms to Cx40 gap junctions expressed in mouse neuro2A (N2A) cells. Junctional currents resembled the action potential morphology but declined by >50% from peak to near-constant plateau values. Kinetics of Cx40 voltage gating were examined at peak voltages > or =100 mV, and decay time constants changed e-fold per 17.6 mV for Vj > +/-40 mV. Junctional conductance recovered during phase 3 repolarization and early diastole to initial values. These phasic changes in junctional conductance were due to rapid decay kinetics, increasing to tens of milliseconds at peak Vj of 130 mV, and the increase in the steady-state conductance curve as Vj returned toward 0 mV. Time-dependent conductance curves for Cx40 were modeled with one inactivation and two recovery Vj-dependent components. There was a temporal correlation between development of conduction delay or block and the inactivation phase of junctional conductance. Likewise, recovery of junctional conductance was coincident with recovery from refractoriness, suggesting that gap junctions may play a role in the genesis and propagation of cardiac arrhythmias.     

Interaction of DDT with the Components of Lobster Nerve Membrane Conductance

The falling phase of action potential of lobster giant axons is markedly prolonged by treatment with DDT, and a plateau phase appears as in cardiac action potentials. Repetitive afterdischarge is very often superimposed on the plateau. Voltage-clamp experiments with the axons treated with DDT and with DDT plus tetrodotoxin or saxitoxin have revealed the following: DDT markedly slows the turning-off process of peak transient current and suppresses the steady-state current. The falling phase of the peak transient current in the DDT-poisoned axon is no longer expressed by a single exponential function as in normal axons, but by two or more exponential functions with much longer time constants. The maximum peak transient conductance is not significantly affected by DDT. DDT did not induce a shift of the curve relating the peak transient conductance to membrane potential along the potential axis. The time to peak transient current and the time for the steady-state current to reach its half-maximum are prolonged by DDT to a small extent. The finding that, under the influence of DDT, the steady-state current starts flowing while the peak transient current is partially maintained supports the hypothesis of two operationally separate ion channels in the nerve membrane.     

The Repolarization Phase of the Cardiac Ventricular Action Potential: A Time-Dependent System of Membrane Conductances

A system for the generation of the repolarization phase of the ventricular action potential is described. The system is based on time-dependent changes in membrane conductance to sodium and potassium ions. However, the changes in conductance during an action potential retain a degree of voltage dependence through the initial conditions which depend on previous depolarizations of the membrane. The equations describing the system were solved with an analog computer and various action potential forms are reproduced. The effects of hyperpolarizing and depolarizing current applied during an action potential are investigated. The changes in shape of an action potential after a change in the rate of stimulation show partial agreement with previous experimental findings. The applicability of time-dependent and voltage-dependent systems for the generation of the repolarization phase of the ventricular action potential is discussed.     

Generation of action potentials in a mathematical model of corticotrophs.

Corticotropin-releasing hormone (CRH) is an important regulator of adrenocorticotropin (ACTH) secretion from pituitary corticotroph cells. The intracellular signaling system that underlies this process involves modulation of voltage-sensitive Ca2+ channel activity, which leads to the generation of Ca2+ action potentials and influx of Ca2+. However, the mechanisms by which Ca2+ channel activity is modulated in corticotrophs are not currently known. We investigated this process in a Hodgkin-Huxley-type mathematical model of corticotroph plasma membrane electrical responses. We found that an increase in the L-type Ca2+ current was sufficient to generate action potentials from a previously resting state of the model. The increase in the L-type current could be elicited by either a shift in the voltage dependence of the current toward more negative potentials, or by an increase in the conductance of the current. Although either of these mechanisms is potentially responsible for the generation of action potentials, previous experimental evidence favors the former mechanism, with the magnitude of the shift required being consistent with the experimental findings. The model also shows that the T-type Ca2+ current plays a role in setting the excitability of the plasma membrane, but does not appear to contribute in a dynamic manner to action potential generation. Inhibition of a K+ conductance that is active at rest also affects the excitability of the plasma membrane.     

Mechano-perception in Chara cells: the influence of salinity and calcium on touch-activated receptor potentials, action potentials and ion transport

This paper investigates the impact of increased salinity on touch-induced receptor and action potentials of Chara internodal cells. We resolved underlying changes in ion transport by current/voltage analysis. In a saline medium with a low Ca(2+) ion concentration [(Ca(2+))(ext)], the cell background conductance significantly increased and proton pump currents declined to negligible levels, depolarizing the membrane potential difference (PD) to the excitation threshold [action potential (AP)(threshold)]. The onset of spontaneous repetitive action potentials further depolarized the PD, activating K(+) outward rectifying (KOR) channels. K(+) efflux was then sustained and irrevocable, and cells were desensitized to touch. However, when [Ca(2+)](ext) was high, the background conductance increased to a lesser extent and proton pump currents were stimulated, establishing a PD narrowly negative to AP(threshold). Cells did not spontaneously fire, but became hypersensitive to touch. Even slight touch stimulus induced an action potential and further repetitive firing. The duration of each excitation was extended when [Ca(2+)](ext) was low. Cell viability was prolonged in the absence of touch stimulus. Chara cells eventually depolarize and die in the saline media, but touch-stimulated and spontaneous excitation accelerates the process in a Ca(2+)-dependent manner. Our results have broad implications for understanding the interactions between mechano-perception and salinity stress in plants.     

Effects of phlorizin and phloretin on passive and dynamic electrical properties in muscle membrane

The effects of phlorizin and phloretin on the cable properties were investigated in frog sartorius muscle by conventional cable analysis. Actions of phloretin on voltage-dependent ionic conductances were also studied by analysis of the phase plane trajectories. Both drugs evoked a significant decrease in specific membrane resistance (Rm) in chloride-containing Ringer's solution. The linear membrane capacitance increased by about 30%. On the contrary, in the presence of the non-penetrating anion, glutamate, a slight increase in Rm was induced by phlorizin. It is suggested that these drugs may increase the chloride conductance in the muscle membrane. Under the effect of phloretin the resting membrane potential remained unchanged but the amplitude of the action potential was lowered and the rate of repolarization was significantly reduced. The rate of depolarization during the "foot" of the action potential and the conduction velocity calculated from the rate constant of depolarization decreased. The maximum Na conductance was not altered by phloretin but K conductance was reduced. The time constant (tau K) reflecting the kinetic properties of K conductance was increased about seven-fold. It is suggested that great importance may be attributed to the dipole properties of these drugs in the actions presented above.     

Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations

The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil,EGTA, W-7, and TFP on the action potential

Summary The cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La³⁺ or verapamil (a calcium channel blocker) or removal of external Ca²⁺ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca²⁺ channel blockers could not be removed by addition of CaCl₂ to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca²⁺ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.     

Direct action of cGMP on the conductance of retinal rod plasma membrane

In order to identify the intracellular transmitter in the phototransduction process in the retinal rod, the action of cGMP, 2',3'cGMP, cAMP, GMP and Ca2+ on the isolated inside-out patches of the plasma membrane of retinal rods of the frog (Rana temporaria) was studied. cGMP applied at the intracellular membrane surface markedly increased the conductance of patches. The action of cGMP took place in the absence of nucleoside triphosphates and, hence, was not mediated by protein phosphorylation. The dependence of cGMP-induced component of conductance on cGMP concentration was S-shaped, with half-saturation within 10-30 microM and a Hill coefficient of about 1.7-1.8. cAMP, 2',3'cGMP, GMP (1 mM) did not exhibit any action on the membrane. Ca2+ did not affect the patch conductance in the absence of cGMP. In the presence of cGMP, lowering Ca2+ concentration from 10(-3) to 10(-8) M decreased the cGMP-dependent component of conductance by 20-30%. The approximate value of the elementary event underlying the cGMP-induced conductance estimated from the magnitude of the variance of the cGMP-induced current is within 100-250 fS. We suppose that the cGMP-activated channels found by us provide the light-sensitive conductance of the rod plasma membrane in vivo and that cGMP is the intracellular transmitter acting in the phototransduction process.     

Permeability Changes Associated with the Action Potential in Procaine-Treated Crayfish Abdominal Muscle Fibers

Permeability changes associated with prolonged action potentials have been analyzed in procaine-treated crayfish abdominal muscle fibers. The effect of external Ca indicates that the increase in membrane conductance observed during the rising phase of the action potential is primarily due to a permeability increase for Ca. A remnant of the permeability increase may cause the succeeding plateau as shown by its high conductance and by the effect of low Mn. A delayed increase in conductance precedes the termination of the plateau phase. This is due to a delayed increase in permeability, probably for K, that is observed when depolarizing electrogenesis is eliminated. High external Ca reduces the action potential duration, the falling phase starting at a higher depolarization. These changes may be related to an earlier onset of the delayed increase in permeability, induced by a larger inside positivity in the presence of higher Ca. No "anomalous rectification" is seen in early or late I-V curves for small depolarizations. Ba may replace Ca in its role in depolarizing electrogenesis, and the first action potential induced in Ba saline has a large overshoot and a long duration. In higher Ba salines, action potentials are greatly prolonged. Long term soaking in Rb-containing or K-free saline also augments and prolongs the action potential. These changes are assumed to be related to depression of the K permeability of the membrane.