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F Elinder

Publications and source records attributed to F Elinder.

At least 19 recordsLinked to original sources
PubMed2005

Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli.

Apoptotic cell death is an essential process in the development of the central nervous system and in the pathogenesis of its degenerative diseases. Efflux of K(+) and Cl(-) ions leads to the shrinkage of the apoptotic cell and facilitates the activation of caspases. Here, we present electrophysiological and immunocytochemical evidences for the activation of a voltage-dependent anion channel (VDAC) in the plasma membrane of neurons undergoing apoptosis. Anti-VDAC antibodies blocked the channel and inhibited the apoptotic process. In nonapoptotic cells, plasma membrane VDAC1 protein can function as a NADH (-ferricyanide) reductase. Opening of VDAC channels in apoptotic cells was associated with an increase in this activity, which was partly blocked by VDAC antibodies. Hence, it appears that there might be a dual role for this protein in the plasma membrane: (1) maintenance of redox homeostasis in normal cells and (2) promotion of anion efflux in apoptotic cells.

Adenosine Triphosphate↗
PubMed2001

Localization of the extracellular end of the voltage sensor S4 in a potassium channel.

The opening and closing of the pore of voltage-gated ion channels is the basis for the nervous impulse. These conformational changes are triggered by the movement of an intrinsic voltage sensor, the fourth transmembrane segment, S4. The central problem of how the movement of S4 is coupled to channel opening and where S4 is located in relation to the pore is still unsolved. Here, we estimate the position of the extracellular end of S4 in the Shaker potassium channel by analyzing the electrostatic effect of introduced charges in the pore-forming motif (S5-S6). We also present a three-dimensional model for all transmembrane segments. Knowledge of this structure is essential for the attempts to understand how voltage opens these channels.

Animals↗
PubMed2001

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.

Animals↗
PubMed2000

A conserved glutamate is important for slow inactivation in K+ channels.

Voltage-gated ion channels undergo slow inactivation during prolonged depolarizations. We investigated the role of a conserved glutamate at the extracellular end of segment 5 (S5) in slow inactivation by mutating it to a cysteine (E418C in Shaker). We could lock the channel in two different conformations by disulfide-linking 418C to two different cysteines, introduced in the Pore-S6 (P-S6) loop. Our results suggest that E418 is normally stabilizing the open conformation of the slow inactivation gate by forming hydrogen bonds with the P-S6 loop. Breaking these bonds allows the P-S6 loop to rotate, which closes the slow inactivation gate. Our results also suggest a mechanism of how the movement of the voltage sensor can induce slow inactivation by destabilizing these bonds.

Amino Acid Sequence↗
PubMed1999

The screw-helical voltage gating of ion channels.

In the voltage-gated ion channels of every animal, whether they are selective for K+, Na+ or Ca2+, the voltage sensors are the S4 transmembrane segments carrying four to eight positive charges always separated by two uncharged residues. It is proposed that they move across the membrane in a screw-helical fashion in a series of three or more steps that each transfer a single electronic charge. The unit steps are stabilized by ion pairing between the mobile positive charges and fixed negative charges, of which there are invariably two located near the inner ends of segments S2 and S3 and a third near the outer end of either S2 or S3. Opening of the channel involves three such steps in each domain.

Amino Acid Sequence↗
PubMed1999

Role of individual surface charges of voltage-gated K channels.

Fixed charges on the extracellular surface of voltage-gated ion channels influence the gating. In previous studies of cloned voltage-gated K channels, we found evidence that the functional surface charges are located on the peptide loop between the fifth transmembrane segment and the pore region (the S5-P loop). In the present study, we determine the role of individual charges of the S5-P loop by correlating primary structure with experimentally calculated surface potentials of the previously investigated channels. The results suggest that contributions to the surface potential at the voltage sensor of the different residues varies in an oscillating pattern, with the first residue of the N-terminal end of the S5-P loop, an absolutely conserved glutamate, contributing most. An analysis yields estimates of the distance between the residues and the voltage sensor, the first N-terminal residue being located at a distance of 5-6 A. To explain the results, a structural hypothesis, comprising an alpha-helical N-terminal end of the S5-P loop, is presented.

Bacterial Proteins↗
PubMed1998

Mechanisms of bupivacaine action on Na+ and K+ channels in myelinated axons of Xenopus laevis.

The local anaesthetic bupivacaine has recently been proposed to inhibit Na+ channels indirectly by making the resting potential less negative. To test this hypothesis we analysed the effects of bupivacaine on voltage and current clamped nodes of Ranvier. Contrary to the hypothesis, the leak current and the resting potential were unaffected. The Na+ and K+ channels were, however, affected at relatively low concentrations (33 microM). Steady-state activation curves were decreased without notable shift effects, whereas the Na+ inactivation curve was decreased and shifted in negative direction. The effect on the Na+ current was tentatively explained by a single-site, state-dependent binding model (Kd = 44 microM), while that on the K+ current was explained by two population-specific mechanisms, one open-state dependent (Kd = 550 microM) and one state independent (Kd = 59 microM). The binding stoichiometry was higher than 1:1 for the main sites of action. In conclusion, bupivacaine exerts its main anaesthetic action on myelinated nerve axons by a direct modification of Na+ channels.

Anesthetics, Local↗
PubMed1998

The functional surface charge density of a fast K channel in the myelinated axon of Xenopus laevis.

The action of Mg2+ on the putative xKv1.1 channel in the myelinated axon of Xenopus laevis was analyzed in voltage clamp experiments. The main effect was a shift in positive direction of the open probability curve (16 mV at 20 mM Mg2+), calculated from measurements of the instantaneous current at Na reversal potential after 50-100 msec steps to different potentials. The shift was measured at an open probability level of 25% to separate it from shifts of other K channel populations in the nodal region. The results could be explained in terms of screening effects on fixed charges located on the surface of the channel protein. Using the Grahame equation the functional charge density was estimated to -0.45 e nm-2. Analyzing this value, together with previously estimated values from other K channels, with reference to the charge of different extracellular loops of the channel protein, we conclude that the loop between the transmembrane S5 segment and the pore forming P segment determines the functional charge density of voltage-gated K channels.

Animals↗
PubMed1998

Divalent cation effects on the Shaker K channel suggest a pentapeptide sequence as determinant of functional surface charge density.

The effects of the divalent cations strontium and magnesium on Shaker K channels expressed in Xenopus oocytes were investigated with a two-electrode voltage-clamp technique. 20 mM of the divalent cation shifted activation (conductance vs. potential), steady-state inactivation and inactivation time constant vs. potential curves 10-11 mV along the potential axis. The results were interpreted in terms of the surface charge theory, and the surface charge density was estimated to be -0.27 e nm-2. A comparison of primary structure data and experimental data from the present and previous studies suggests that the first five residues on the extracellular loop between transmembrane segment 5 and the pore region constitutes the functional surface charges. The results further suggest that the surface charge density plays an important role in controlling the activation voltage range.

Animals↗
PubMed1998

On the slowly rising phase of the sodium gating current in the squid giant axon.

High-resolution records of the sodium gating current in the squid giant axon demonstrate the existence of a slowly rising phase that is first apparent at pulse potentials slightly below zero, and becomes increasingly pronounced at more positive potentials. At +80 mV the current reaches its peak with a delay of 30 microseconds at 10 degrees C. It is suggested that this current is generated by the first two steps labelled R-->P and P-->A in the S4 units of all four domains of the series-parallel gating system, activating the channel before its opening by the third steps A-->B in domains I, II and III in conjunction with hydration. The kinetics of the slowly rising phase can only be explained by the incorporation of an appropriate degree of voltage-dependent cooperativity between the S4 voltage-sensors for their two initial transitions.

Animals↗
PubMed1998

Modelling the activation, opening, inactivation and reopening of the voltage-gated sodium channel.

A model of the voltage-gated sodium channel is put forward suggesting that the four S4 voltage-sensors behave as screw-helices making a series of discrete transitions that carry one elementary charge for each notch of the screw helix. After the channel has been activated by the first two steps R in equilibrium with P in equilibrium with A in all four domains, followed by a voltage-independent rearrangement, it is opened by a third cooperative step A in equilibrium with B in domains I, II and III in conjunction with hydration. Inactivation is a voltage-dependent process controlled by the third step A in equilibrium with I in sensor IVS4, and the closing of the channel is brought about its dehydration. From the inactivated steady state the channel may be reopened by a fourth step, I in equilibrium with C in sensor IVS4 and rehydration. The computed kinetics of the model are shown to conform closely with those observed experimentally.

Animals↗
PubMed1997

Tail currents in the myelinated axon of Xenopus laevis suggest a two-open-state Na channel.

Na tail currents in the myelinated axon of Xenopus laevis were measured at -70 mV after steps to -10 mV. The tail currents were biexponential, comprising a fast and a slow component. The time constant of the slow tail component, analyzed in the time window 0.35-0.50 ms, was independent of step duration, and had a value of 0.23 ms. The amplitude, extrapolated back to time 0, varied, however, with step duration. It reached a peak after 0.7 ms and inactivated relatively slowly (at 2.1 ms the absolute value was reduced by approximately 30%). The amplitude of the fast component, estimated by subtracting the amplitude of the slow component from the calculated total tail current amplitude, reached a peak (three times larger than that of the slow component) after 0.5 ms and inactivated relatively fast (at 2.1 ms it was reduced by approximately 65%). The results were explained by a novel Na channel model, comprising two open states bifurcating from a common closed state and with separate inactivating pathways. A voltage-regulated use of the two pathways explains a number of findings reported in the literature.

Animals↗
PubMed1996

Surface Charges of K channels. Effects of strontium on five cloned channels expressed in Xenopus oocytes.

The effects of strontium (Sr2+; 7-50 mM) on five different cloned rat K channels (Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv3.4), expressed in oocytes of Xenopus laevis, were investigated with a two-electrode voltage clamp technique. The main effect was a shift of the Gk(V) curve along the potential axis, different in size for the different channels. Kv1.1 was shifted most and Kv3.4 least, 21 and 8 mV, respectively, at 50 mM. The effect was interpreted in terms of screening of fixed surface charges. The estimated charge densities ranged from -0.37 (Kv1.1) to -0.11 (Kv3.4) e nm-2 and showed good correlation with the total net charge of the extracellularly located amino acid residues of the channel as well as with the charge of a specific region (the loop between the S5 segment and the pore forming segment). The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.

Amino Acid Sequence↗
PubMed1994

Effects of gadolinium on ion channels in the myelinated axon of Xenopus laevis: four sites of action.

The action of gadolinium (Gd3+) on ion currents in myelinated axons of Xenopus laevis was investigated with the voltage clamp technique. The analysis revealed the following effects. (i) The potential-dependent parameters of both Na and K channels were shifted. The shift was equally large for activation, inactivation, and activation time constant curves (+9 mV for 100 microM Gd3+). The effects could be explained by screening of fixed surface charges at a density of -1.2 e nm-2. (ii) The rate of gating for both Na and K channels was reduced more than predicted from the shift. This effect could be quantified as a scaling (by a factor 3 and 5 respectively at 100 microM Gd3+) of the activation time constant curves. (iii) An activation- and inactivation-independent block of both Na and K channels, obeying 1:1 stoichiometry with a Kd value of about 70 microM potential-independent block of leakage current, obeying 1:2 stoichiometry with a Kd value of 600 microM. (iv) The analysis suggests separate binding sites for the effects, comprising high affinity modulatory and blocking sites on the channel proteins and low affinity receptors on phospholipids, associated with the effect on the leakage current.

Animals↗
PubMed1994

The modulatory site for the action of gadolinium on surface charges and channel gating.

The gadolinium (Gd3+)-induced shift of potential dependence and modulated gating of Na and K channels were analyzed. In a previous investigation, we explained the shift in terms of pure screening (no binding) of fixed surface charges and the modulation by binding to modulatory sites on the channels. In the present paper, we have extended this model by including effects on the charge density of Gd3+ binding to the modulatory sites. From fitting the extended model to experimental data, the charge density was estimated to be -0.6 e nm-2, and the Gd(3+)-induced charge change to be +0.15 e nm-2, and the maximal scaling factor to be 7.5 for both Na and K channels. Intrinsic KD values for binding to the K and Na channels were estimated to be 140 and 380 mM, respectively. Estimations of the extracellular charge density, from primary structures of cloned channels, were found to be in agreement with estimations based on the present model. The modulatory site was suggested to be located at the cluster of negatively charged residues between the fifth transmembrane segment (S5) and the pore-forming region for both Na and K channels. These suggestions imply several testable predictions about different K channels.

Animals↗
PubMed1992

High concentrations of the neuroleptic remoxipride block voltage-activated Na+ channels in central and peripheral nerve membranes.

The actions of the neuroleptic compounds remoxipride, haloperidol and (-)-sulpiride on Na+ and K+ ion current flow were examined in rat CNS and frog PNS, using 86Rb+ ion flux and voltage-clamp techniques, respectively. By combining veratridine and high K(+)-evoked 86Rb+ efflux, it was determined that remoxipride blocked Na+ current flow in a concentration-dependent fashion in tissue pieces from either cerebral cortex or striatum (IC50 approximately 20 microM), leaving K+ current flow virtually unaffected. Similarly, haloperidol concentration dependently blocked Na+ current flow in both tissues (IC50 approximately 50 microM). (-)-Sulpiride did not have a significant effect. Direct actions of the compounds on voltage-gated Na+ and K+ channels were determined in voltage-clamp experiments. The findings confirmed the results of the ion flux measurements in that remoxipride (Kd approximately 300 microM) and haloperidol (Kd approximately 1.5 microM) reduced mainly the Na+ current, having little effect on the K+ current, whereas (-)-sulpiride did not have a measurable action. The relatively high concentrations of remoxipride or haloperidol needed to alter the Na+ current makes it unlikely that these actions are of importance at clinically relevant doses.

Animals↗
PubMed1992

Mechanisms of propofol action on ion currents in the myelinated axon of Xenopus laevis.

The effect of the intravenous anaesthetic, propofol (2,6-diisopropylphenol), was investigated on frog myelinated axons under voltage-clamp conditions. The effect, in the concentration range 60 microM to 10 mM, was a combination of (i) a negative shift of the steady state activation and inactivation curves for both Na+ and K+ currents (INa,IK), (ii) a voltage-independent block of INa, but not of IK, and (iii) a slowed time course of IK activation. The shift was dose-dependent and, at 1 mM, about -10 mV for the activation and -16 mV for the inactivation curves. The voltage-independent INa block showed 1:1 stoichiometry and 50% reduction at 2.7 mM. The slowed IK activation showed saturation at 1 mM with a doubled time to half steady state value. All the effects were only partially reversible and showed a complex time course at application and washing. The shift of potential dependence may be explained by a general effect on the membrane electric field. The findings suggest effects directly on channel proteins as well as on membrane lipids.

Anesthetics↗