Fredrik Elinder

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.

Functionalised ZnO-nanorod-based selective electrochemical sensor for intracellular glucose

In this article, we report a functionalised ZnO-nanorod-based selective electrochemical sensor for intracellular glucose. To adjust the sensor for intracellular glucose measurements, we grew hexagonal ZnO nanorods on the tip of a silver-covered borosilicate glass capillary (0.7 microm diameter) and coated them with the enzyme glucose oxidase. The enzyme-coated ZnO nanorods exhibited a glucose-dependent electrochemical potential difference versus an Ag/AgCl reference microelectrode. The potential difference was linear over the concentration range of interest (0.5-1000 microM). The measured glucose concentration in human adipocytes or frog oocytes using our ZnO-nanorod sensor was consistent with values of glucose concentration reported in the literature; furthermore, the sensor was able to show that insulin increased the intracellular glucose concentration. This nanoelectrode device demonstrates a simple technique to measure intracellular glucose concentration.

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.

Tracking a complete voltage-sensor cycle with metal-ion bridges

Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 Å along its axis to open the channel with a 310 helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states.

Hysteresis in the Voltage Dependence of HCN Channels: Conversion between Two Modes Affects Pacemaker Properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (τ ≈ 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.

Metal ion effects on ion channel gating

Metal ions affect ion channels either by blocking the current or by modifying the gating. In the present review we analyse the effects on the gating of voltage-gated channels. We show that the effects can be understood in terms of three main mechanisms. Mechanism A assumes screening of fixed surface charges. Mechanism B assumes binding to fixed charges and an associated electrostatic modification of the voltage sensor. Mechanism C assumes binding and an associated non electrostatic modification of the gating. To quantify the non-electrostatic effect we introduced a slowing factor, A. A fourth mechanism (D) is binding to the pore with a consequent pore block, and could be a special case of Mechanisms B or C. A further classification considers whether the metal ion affects a single site or multiple sites. Analysing the properties of these mechanisms and the vast number of studies of metal ion effects on different voltage-gated on channels we conclude that group 2 ions mainly affect channels by classical screening (a version of Mechanism A). The transition metals and the Zn group ions mainly bind to the channel and electrostatically modify the gating (Mechanism B), causing larger shifts of the steady-state parameters than the group 2 ions, but also different shifts of activation and deactivation curves. The lanthanides mainly bind to the channel and both electrostatically and non-electrostatically modify the gating (Mechanisms B and C). With the exception of the ether-à-go-go-like channels, most channel types show remarkably similar ion-specific sensitivities.

Lipoelectric Modification of Ion Channel Voltage Gating by Polyunsaturated Fatty Acids

Polyunsaturated fatty acids (PUFAs) have beneficial effects on epileptic seizures and cardiac arrhythmia. We report that omega-3 and omega-6 all-cis-PUFAs affected the voltage dependence of the Shaker K channel by shifting the conductance versus voltage and the gating charge versus voltage curves in negative direction along the voltage axis. Uncharged methyl esters of the PUFAs did not affect the voltage dependence, whereas changes of pH and charge mutations on the channel surface affected the size of the shifts. This suggests an electrostatic effect on the channels voltage sensors. Monounsaturated and saturated fatty acids, as well as trans-PUFAs did not affect the voltage dependence. This suggests that fatty acid tails with two or more cis double bonds are required to place the negative carboxylate charge of the PUFA in a position to affect the channels voltage dependence. We propose that charged lipophilic compounds could play a role in regulating neuronal excitability by electrostatically affecting the channels voltage sensor. We believe this provides a new approach for pharmacological treatment that is voltage sensor pharmacology.

Molecular Movement of the Voltage Sensor in a K Channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 Å) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 Å) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.

Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells

microRNAs (miRNAs) are small non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis and proliferation. Several miRNAs display defective expression patterns in human tumors with the consequent alteration of target oncogene or tumor suppressor genes. Many of these miRNAs modulate the major proliferation pathways through direct interaction with critical regulators such as RAS, PI3K/PTEN or ABL, as well as members of the retinoblastoma pathway, Cyclin-CDK complexes or cell cycle inhibitors of the INK4 or Cip/Kip families. A complex interplay between miRNAs and MYC or E2F family members also exists to modulate cell cycle-dependent transcription during normal or tumoral proliferation. The ability of miRNAs to modulate these proliferation pathways may have relevant implications not only in physiological or developmental processes but also in tumor progression or cancer therapy.

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.