Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region. Carbonyl stretch modes of specific amino acid residues helped to elucidate structural changes in the retinal Schiff base moiety, including the protonation and deprotonation of D116, which is crucial for a deeper understanding of the mechanistic features in the photocycle of KR2.

A mammalian retinal bipolar cell uses both graded changes in membrane voltage and all-or-nothing Na+ spikes to encode light.

Barlow (1953) studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na(+) spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na(+) current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1-3 all-or-nothing action potentials that attain a peak amplitude of -10 to -20 mV (peak width at half-height equals 2-3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b cells generate both graded and spiking responses. Cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells costratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions.

Effect of superoxide derived from lucifer yellow CH on voltage-gated currents of mouse taste bud cells.

Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.

Molecular basis for function in sodium channels.

Na+ channels earned their unique role in excitable cells because of two functional properties, finely honed by evolution. The first is their exquisite sensitivity to small changes of membrane potential: a depolarization of only 10 mV can increase open probability by as much as two orders of magnitude. The second is the rapidity with which they respond to changes of membrane potential: their gates begin to open tens of microseconds after a depolarization. These features are built into two sets of moving parts: voltage sensors that respond directly to changes of membrane potential, and gates that open and close in response to voltage sensor movement. We have explored these movements using a combination of electrophysiology, site-directed mutagenesis, cysteine accessibility scanning and photoactivated cross-linking using a bifunctional cysteine reagent. The main voltage sensors of Na+ channels are four homologous S4 segments, each of which has a unique functional role. These transmembrane segments are almost completely surrounded by hydrophilic crevices. The membrane electric field moves these positively charged helices through a short, hydrophobic 'gating pore'. The minimum contact between an S4 segment and its gating pore insure that a small movement can rapidly move several of its charged residues across the electric field.

Modulation of sodium currents in rat sensory neurons by nucleotides.

The effects of various nucleotides on the fast tetrodotoxin-sensitive (f-TTX-S) and the slow tetrodotoxin-resistant (s-TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were investigated using the patch-clamp technique. Nucleoside triphosphates (NTPs; ATP, GTP, UTP and CTP) and nucleoside diphosphates (NDPs; ADP, GDP, UDP and CDP) decreased f-TTX-S current, whereas they increased s-TTX-R current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. NTPs and NDPs shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction in both types of sodium currents. Most of them also increased the maximum conductance of s-TTX-R current. ITP, a derivative of ribonucleotide, and dTTP, a deoxyribonucleotide, modulated both types of sodium currents similarly to NTPs and NDPs. However, nucleoside monophosphates (NMPs; AMP, GMP, UMP and CMP) and adenosine had little or no effect on either type of sodium current. Therefore, it seems that nucleotides, regardless of the kind of base, should have two or more phosphates to be able to modulate sodium currents in DRG neurons. Extracellular nucleotides with di- or tri-phosphates would influence the perception by modulating sodium currents in sensory neurons. Particularly, the increase of the maximum conductance and the hyperpolarizing shift of the conductance-voltage relationship of s-TTX-R sodium current would result in an intensified nociception.

Effect of a 0.5-T static magnetic field on conduction in guinea pig spinal cord.

Compound-evoked potentials were recorded from excised adult guinea pig spinal cords before, during, and following exposure to a 0.5-T static magnetic field (SMF). There was no change in response latency during exposure but there was a small, statistically significant, decrease in amplitude. Maximum effect was evident 1 to 2 min after the field was turned on with return to baseline within 1 min after the field was turned off. These results may be explained by a conduction block in the small axon subpopulation due to the effect of static magnetic fields on voltage-activated sodium channels. The relative selectivity of the field is believed to occur because of the relatively greater number of sodium channels present in smaller axons.

Effects of X-irradiation on the calcium channel of the mouse oocyte.

Effects of a single whole-body X-irradiation (0.2-100 Gy) were studied on Ca2+ channels in mature mouse oocytes using the two-electrode voltage-clamp method. No significant changes were observed in the electrophysiological properties of oocytes when the dose of X-rays was smaller than 10 Gy. However, the following changes, dependent on the dose of X-rays and on the time after irradiation, were observed with higher doses. 1) The resting potential and the input resistance of the oocyte were slightly reduced. 2) Inward Ca2+ current was reduced in amplitude but the shape of the I-V (current-to-voltage) relations were preserved. 3) Both the activation and the inactivation processes of the Ca2+ current became slower. These changes in the kinetics of the Ca2+ channel were observed even when no appreciable change was detected in the amplitude of the Ca2+ current. 4) The steady-state inactivation curve of the Ca2+ current was shifted in a depolarizing direction and the slope of the curve became steeper. These data indicate that high doses of X-rays can affect Ca2+ channels in mouse oocytes and that the kinetics of the Ca2+ channel are more susceptible to irradiation than the amplitude of the Ca2+ current.

[Effects of microwave irradiation on ATPase activity and voltage dependent ion channel of rat hippocampus cell membrane].

OBJECTIVE: To study the effect of microwave irradiation on hippocampus cell. METHOD: Changes of ATPase activity and voltage dependent ion channel of hippocampus cell membrane were observed in mice exposed to 2 450 MHz microwave irradiation of 10 mW/cm2 from a physical therapy machine. Histochemical method and patch clamp method were used to determine the activity of Na+, K(+)-ATPase, Ca2+, Mg(2+)-ATPase and voltage dependent Na+, K+, Ca2+ channels respectively. RESULT: 1) Na+, K(+)-ATPase activity of microwave irradiated mice showed no significant change as compared with the control, but the activity of Ca2+, Mg(2+)-ATPase decreased significantly (P< 0.05); 2) In microwave irradiated mice, Na+, K+, Ca2+, current inducement rate in hippocampus neuron decreased significantly, the membrane voltage of Na+ current peak shifted to depolarization, and the attenuation rate of Na+ current and current A inducement rate decreased significantly as compared with control mice. CONCLUSION: Irradiation of 2 450 MHz microwave at a doze of 10 mW/cm2 was not fatal to mice hippocampus cell. But Ca2+, Mg(2+)-ATPase activity of hippocampal cell membrane and voltage dependent Na+, K+, Ca2+ ion channel of hippocampal nervous were affected which would affect study and memory.

980-nm infrared laser modulation of sodium channel kinetics in a neuron cell linearly mediated by photothermal effect.

Photothermal effect (PE) plays a major role in the near-infrared laser interaction with biological tissue. But, quite few interactions can be quantitatively depicted. Here, a two-step model is proposed to describe a 980-nm infrared laser interaction with neuron cell in vitro. First, the laser-induced temperature rises in the cell surrounding area were measured by using an open pipette method and also calculated by solving the heat conduction equation. Second, we recorded the modifications on sodium (Na) channel current in neuron cells directly by using a patch clamp to synchronize the 980-nm laser irradiation and obtained how the electrophysiological function of neuron cells respond to the temperature rise. Then, the activation time constants, τ(m), were extracted by fitting the sodium currents with the Hodgkin-Huxley model. The infrared laser modulation effect on sodium currents kinetics was examined by taking a ratio between the time constants with and without the laser irradiations. The analysis revealed that the averaged ratio at a specific laser exposure could be well related to the temperature properties of the Na channel protein. These results proved that the modulation of sodium current kinetics of a neuron cell in vitro by 980-nm laser with different-irradiation levels was linearly mediated corresponding to the laser-induced PE.

Initiation and blocking of the action potential in an axon in weak ultrasonic or microwave fields.

In this paper, we analyze the effect of the redistribution of the transmembrane ion channels in an axon caused by longitudinal acoustic vibrations of the membrane. These oscillations can be excited by an external source of ultrasound and weak microwave radiation interacting with the charges sitting on the surface of the lipid membrane. It is shown, using the Hodgkin-Huxley model of the axon, that the density redistribution of transmembrane sodium channels may reduce the threshold of the action potential, up to its spontaneous initiation. At the significant redistribution of sodium channels in the membrane, the rarefaction zones of the transmembrane channel density are formed, blocking the propagation of the action potential. Blocking the action potential propagation along the axon is shown to cause anesthesia in the example case of a squid axon. Various approaches to experimental observation of the effects considered in this paper are discussed.

Can electromagnetic radiations induce changes in the kinetics of voltage-dependent ion channels?

Ion channels are protein molecules, which can assume distinct open and closed conformational states, a phenomenon termed ion channel kinetics. The transitions from one state to another depend on the potential energy barrier that separates those two states. Therefore, it is rational to suppose that electromagnetic waves could interact with this barrier and induce changes in the rate transitions of this kinetic process. Our aim is to answer the question: can electromagnetic radiations induce changes in the kinetics of voltage-dependent ion channels? We simulated the effects of the low and high frequency electromagnetic waves on the sodium and potassium channels of the giant axon of Loligo. The key parameter measured was the fractional open time (fv), because it reflects the voltage dependence of the kinetics of channels. The electromagnetic radiations induced the following changes in the kinetics of the potassium and sodium channels: i/ low frequency waves kept the potassium channel 50% of the time open independent on the mean voltage applied through the membrane; ii/ a gradual inhibition of the inactivation on the sodium channel, when the amplitudes of the low frequency waves were increased; iii/ high frequency waves on the potassium channel, decreased both Vo (voltage in which the channel stays 50% open) and the steepness of fv (d fv/dV) as the amplitudes of the waves increased, and iv/ high frequency and low amplitude radiations on the sodium channel decreased the maximum value of fv (in relation to control), while high amplitudes increased this value. In conclusion, high and low frequency electromagnetic radiations were able to change the kinetics of the potassium and sodium channels in a squid giant axon model.

Effect of a 125 mT static magnetic field on the kinetics of voltage activated Na+ channels in GH3 cells.

Voltage activated Na(+) channels were examined in GH3 cells, using the whole cell patch clamp method. Channel currents were recorded before, during, and after a 150 s exposure to a 125 mT static magnetic field. There was a slight shift in the current-voltage relationship and a less than 5% reduction in peak current during magnetic field exposure. More pronounced, however, was an increase in the activation time constant, tau(m), during and for at least 100 s following exposure to the field. This change in tau(m) was seen primarily at lower activation voltages. No change was noted in the inactivation time constant, tau(h). Changes were clearly temperature dependent, being evident only at and above 35 degrees C. These findings are consistent with the hypothesis that reorientation of diamagnetic anisotropic molecules in the cell membrane are capable of distorting imbedded ion channels sufficiently to alter their function. The temperature dependence of this phenomenon is probably due to the greater ease with which a liquid crystal membrane can be deformed.

Fast and slow inactivation of sodium channels: effects of photodynamic modification by methylene blue.

Illumination of crayfish giant axons, during internal perfusion with 0.5 mM methylene blue (MB), produces photodynamic effects that include (i) reduction in total sodium conductance, (ii) shifting of the steady-state inactivation curve to the right along the voltage axis, (iii) reduction in the effective valence of steady-state inactivation and, (iv) potentially complete removal of fast inactivation. Additionally, the two kinetic components of fast inactivation in crayfish axons are differentially affected by MB+light. The intercept of the faster component (tau h1) is selectively reduced at shorter MB+light exposure times. Neither tau h1 nor the slower (tau h2) process was protected from MB+light by prior steady-state inactivation of sodium channels. However, carotenoids provide differing degrees of protection against each of the photodynamic actions listed above, suggesting that the four major effects of MB+light are mediated by changes occurring within different regions of the sodium channel molecule.

Effects of extremely-low-frequency electromagnetic fields on ion transport in several mammalian cells.

We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca2+, Na+, K+, and H+) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca2+ concentration ([Ca2+]i), the intracellular pH (pHi), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100-2000 microT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20-1200 microT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12-32% increase of the uptake of 42K within a window at 14.5-15.5 Hz and 100-200 microT (rms), which was found in U937 and Ehrlich cells but not in the other cell types.

[Postradiation changes of active ion transport systems of the CNS. The effect of superlethal doses of ionizing radiation on the Na,K pump in surviving brain slices]. / Postradiatsionnye izmeneniia v sistemakh aktivnogo transporta ionov v TsNS. Vliianie sverkletal'nykh doz ioniziruiushchei radiatsii na Na,K-nasos v perezhivaiushchikh srezakh mozga.

Active potassium ion transport in surviving sections of rat brain was irreversibly inhibited 6 min, 1 h and 6 h following whole-body single X irradiation. At the same time, the accumulation of products of lipid peroxidation and phospholipase hydrolysis was followed up during the development of radiation pathology. The relationship was noted between the postirradiation changes in the physicochemical status of lipids of the membrane and the impairment of its transport function.

Lucifer Yellow slows voltage-gated Na+ current inactivation in a light-dependent manner in mice.

Lucifer Yellow CH (LY), a membrane-impermeant fluorescent dye, has been used in electro-physiological studies to visualize cell morphology, with little concern about its pharmacological effects. We investigated its effects on TTX-sensitive voltage-gated Na+ channels in mouse taste bud cells and hippocampal neurons under voltage-damp conditions. LY applied inside cells irreversibly slowed the inactivation of Na+ currents upon exposure to light of usual intensities. The inactivation time constant of Na+ currents elicited by a depolarization to -15 mV was increased by fourfold after a 5 min exposure to halogen light of 3200 Ix at source (3200 Ix light), and sevenfold after a 1-min exposure to 12,000 Ix light. A fraction of the Na+ current became non-inactivating following the exposure. The non-inactivating current was approximately 20 % of the peak total Na+ current after a 5 min exposure to 3200 Ix light, and approximately 30 % after a 1 min exposure to 12,000 Ix light. Light-exposed LY shifted slightly the current-voltage relationship of the peak Na+ current and of the steady-state inactivation curve, in the depolarizing direction. A similar light-dependent decrease in kinetics occurred in whole-cell Na+ currents of cultured mouse hippocampal neurones. Single-channel recordings showed that exposure to 6500 Ix light for 3 min increased the mean open time of Na+channels from 1.4 ms to 2.4 ms without changing the elementary conductance. The pre-incubation of taste bud cells with 1 mM dithiothreitoL a scavenger of radical species, blocked these LY effects. These results suggest that light-exposed LY yields radical species that modify Na+ channels.

[The action of ionizing radiation on neuronal function in the edible snail. Phospholipase A2 and the Na+ and K+ transport systems]. / Deistvie ioniziruiushchei radiatsii na funktsional'noe sostoianie neironov vinogradnoi ulitki. Fosfolipaza A2 i sistemy transporta Na+ i K+.

A study was made of the influence of A2 phospholipase on 22Na release from cells of nerve ganglia of edible snail. The treatment of nerve ganglia with A2 phospholipase inhibits Na, K-pump of neuronal membranes and does not exert a substantial effects on Na/Ca metabolism. There is a similarity between the effects of ionizing radiation and A2 phospholipase on the release of 22Na from cells.

Modifications of human cardiac sodium channel gating by UVA light.

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.

Induction of pseudo-periodic oscillation in voltage-gated sodium channel properties is dependent on the duration of prolonged depolarization.

The neuronal voltage-gated sodium channels play a vital role in the action potential waveform shaping and propagation. Here, we report the effects of prolonged depolarization (1-160 s) on the detailed kinetics of activation, fast inactivation and recovery from slow inactivation in the rNa(v)1.2a voltage-gated sodium channel alpha-subunit expressed in Chinese hamster ovary (CHO) cells. Wavelet analysis revealed that the duration and amplitude of a prolonged sustained depolarization altered all the steady state and kinetic parameters of the channel in a pseudo-oscillatory fashion with time-variable period and amplitude, often superimposed on a linear trend. The half steady state activation potential showed a reversible depolarizing shift of 5-10 mV with duration of prolonged depolarization, while half steady state inactivation potential showed a hyperpolarizing shift of 43-55 mV. The time periods for most of the parameters relating to activation and fast and slow inactivation, lie close to 28-30 s, suggesting coupling of these kinetic processes through an oscillatory mechanism. Co-expression of the beta1-subunit affected the time periods of oscillation (close to 22 s for alpha + beta1) in steady state activation parameters. Application of a pulse protocol that mimicked paroxysmal depolarizing shift (PDS), a kind of depolarization seen in epileptic discharges, instead of a sustained depolarization, also caused oscillatory behaviour in the rNav1.2a alpha-subunit. This inherent pseudo-oscillatory mechanism may regulate excitability of the neurons, account for the epileptic discharges and subthreshold membrane potential oscillation and offer a molecular memory mechanism intrinsic to the neurons, independent of synaptic plasticity.

Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy.

The persistent sodium current is a common target of anti-epileptic drugs and contributes to burst firing. Intrinsically burst firing subicular neurons are involved in the generation and spread of epileptic activity. We measured whole-cell sodium currents in pyramidal neurons isolated from the subiculum resected in drug-resistant epileptic patients and in rats. In half of the cells from both patients and rats, the sodium current inactivated within 500 ms at -30 mV. Others displayed a tetrodotoxin-sensitive slowly or non-inactivating sodium current of up to 53% of the total sodium current amplitude. Compared with the transient sodium current in the same cells, this persistent sodium current activated with normal kinetics but its voltage-dependent activation occurred 7 mV more hyperpolarized. Depolarizing voltage steps that lasted 10 s completely inactivated the persistent sodium current. Its voltage dependence did not differ from that of the transient sodium current but its slope was less steep. The voltage dependence and kinetics of the persistent sodium current in cells from patients were not different from that in subicular cells from rats. The current density and the relative amplitude contribution were 3-4 times greater in neurons from drug-resistant epilepsy patients. The abundant presence of persistent sodium current in half of the subicular neurons could lead to a larger number of neurons with intrinsic burst firing. The extraordinarily large amplitude of the persistent sodium current in this subset of subicular neurons might explain why these patients are susceptible to seizures and hard to treat pharmacologically.