Sara I. Börjesson

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.

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.

An electrostatic potassium channel opener targeting the final voltage sensor transition.

Free polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of voltage-gated ion channels. As an important consequence thereof, PUFAs can suppress epileptic seizures and cardiac arrhythmia. However, molecular details for the interaction between PUFA and ion channels are not well understood. In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect. Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect. Because different voltage-gated K channels have different charge profiles, this implies channel-specific PUFA effects. The identified site and the pharmacological mechanism will potentially be very useful in future drug design of small-molecule compounds specifically targeting neuronal and cardiac excitability.

Electrostatic Tuning of Cellular Excitability

Voltage-gated ion channels regulate the electric activity of excitable tissues, such as the heart and brain. Therefore, treatment for conditions of disturbed excitability is often based on drugs that target ion channels. In this study of a voltage-gated K channel, we propose what we believe to be a novel pharmacological mechanism for how to regulate channel activity. Charged lipophilic substances can tune channel opening, and consequently excitability, by an electrostatic interaction with the channels voltage sensors. The direction of the effect depends on the charge of the substance. This was shown by three compounds sharing an arachidonyl backbone but bearing different charge: arachidonic acid, methyl arachidonate, and arachidonyl amine. Computer simulations of membrane excitability showed that small changes in the voltage dependence of Na and K channels have prominent impact on excitability and the tendency for repetitive firing. For instance, a shift in the voltage dependence of a K channel with -5 or +5 mV corresponds to a threefold increase or decrease in K channel density, respectively. We suggest that electrostatic tuning of ion channel activity constitutes a novel and powerful pharmacological approach with which to affect cellular excitability.

Functionalized zinc oxide nanorod with ionophore-membrane coating as an intracellular Ca2+ selective sensor

The tip of a borosilicate glass capillary with functionalized hexagonal ZnO nanorods was used to make a sensitive electrochemical intracellular Ca2+ sensor. To adjust the sensor for Ca2+ measuremen ...

Polyunsaturated fatty acids and cerebrospinal fluid from children on the ketogenic diet open a voltage-gated K channel: a putative mechanism of antiseizure action.

PURPOSE Many children with epilepsy do not satisfactorily respond to conventional pharmacological therapy, but to the ketogenic diet, a high-fat, low-carbohydrate diet. This diet increases the concentrations of ketone bodies and polyunsaturated fatty acids (PUFAs) in cerebrospinal fluid (CSF) and plasma. However, its anticonvulsant mechanism is not known. METHODS To investigate the mechanism by which the diet protects against seizures, we studied the effects of several PUFAs (docosahexaenoic acid, eicosapentaenoic acid, and linoleic acid), ketone bodies (beta-hydroxybuturic acid and acetoacetic acid), and CSF from patients on the ketogenic diet on the voltage-gated Shaker K channel expressed in Xenopus oocytes. RESULTS We found that PUFAs at concentrations down to 21microM clearly increased the K current by shifting the conductance versus voltage curve in negative direction along the voltage axis. CSF from patients on the ketogenic diet has similar but smaller effects. In contrast, high concentrations (1-5mM) of ketone bodies did not affect the K current. Computer simulations showed that the observed shifts for clinically relevant concentrations of PUFAs, and CSF from patients could effectively impair repetitive firing. CONCLUSIONS These data suggest that the ketogenic diet could prevent epileptic seizures by PUFA-induced openings of voltage-gated K channels.

Dampening of hyperexcitability in CA1 pyramidal neurons by polyunsaturated fatty acids acting on voltage-gated ion channels.

A ketogenic diet is an alternative treatment of epilepsy in infants. The diet, rich in fat and low in carbohydrates, elevates the level of polyunsaturated fatty acids (PUFAs) in plasma. These substances have therefore been suggested to contribute to the anticonvulsive effect of the diet. PUFAs modulate the properties of a range of ion channels, including K and Na channels, and it has been hypothesized that these changes may be part of a mechanistic explanation of the ketogenic diet. Using computational modelling, we here study how experimentally observed PUFA-induced changes of ion channel activity affect neuronal excitability in CA1, in particular responses to synaptic input of high synchronicity. The PUFA effects were studied in two pathological models of cellular hyperexcitability associated with epileptogenesis. We found that experimentally derived PUFA modulation of the A-type K (KA) channel, but not the delayed-rectifier K channel, restored healthy excitability by selectively reducing the response to inputs of high synchronicity. We also found that PUFA modulation of the transient Na channel was effective in this respect if the channels steady-state inactivation was selectively affected. Furthermore, PUFA-induced hyperpolarization of the resting membrane potential was an effective approach to prevent hyperexcitability. When the combined effect of PUFA on the KA channel, the Na channel, and the resting membrane potential, was simulated, a lower concentration of PUFA was needed to restore healthy excitability. We therefore propose that one explanation of the beneficial effect of PUFAs lies in its simultaneous action on a range of ion-channel targets. Furthermore, this work suggests that a pharmacological cocktail acting on the voltage dependence of the Na-channel inactivation, the voltage dependences of KA channels, and the resting potential can be an effective treatment of epilepsy.

The free energy barrier for arginine gating charge translation is altered by mutations in the voltage sensor domain.

The gating of voltage-gated ion channels is controlled by the arginine-rich S4 helix of the voltage-sensor domain moving in response to an external potential. Recent studies have suggested that S4 moves in three to four steps to open the conducting pore, thus visiting several intermediate conformations during gating. However, the exact conformational changes are not known in detail. For instance, it has been suggested that there is a local rotation in the helix corresponding to short segments of a 3-helix moving along S4 during opening and closing. Here, we have explored the energetics of the transition between the fully open state (based on the X-ray structure) and the first intermediate state towards channel closing (C), modeled from experimental constraints. We show that conformations within 3 Å of the X-ray structure are obtained in simulations starting from the C model, and directly observe the previously suggested sliding 3-helix region in S4. Through systematic free energy calculations, we show that the C state is a stable intermediate conformation and determine free energy profiles for moving between the states without constraints. Mutations indicate several residues in a narrow hydrophobic band in the voltage sensor contribute to the barrier between the open and C states, with F233 in the S2 helix having the largest influence. Substitution for smaller amino acids reduces the transition cost, while introduction of a larger ring increases it, largely confirming experimental activation shift results. There is a systematic correlation between the local aromatic ring rotation, the arginine barrier crossing, and the corresponding relative free energy. In particular, it appears to be more advantageous for the F233 side chain to rotate towards the extracellular side when arginines cross the hydrophobic region.

Intracellular K+ Concentration Decrease Is Not Obligatory for Apoptosis

Background: Decrease in intracellular K+ is an early event during apoptosis. Results: Prevention of intracellular K+ decrease did not prevent activation of caspase-3. Conclusion: A decrease of K+ is not obligatory for cell death in Xenopus oocytes. Significance: The role of monovalent ions is important for understanding the mechanisms behind cell death. K+ efflux is observed as an early event in the apoptotic process in various cell types. Loss of intracellular K+ and subsequent reduction in ionic strength are suggested to release the inhibition of proapoptotic caspases. In this work, a new K+-specific microelectrode was used to study possible alterations in intracellular K+ in Xenopus laevis oocytes during chemically induced apoptosis. The accuracy of the microelectrode to detect changes in intracellular K+ was verified with parallel electrophysiological measurements. In concordance with previous studies on other cell types, apoptotic stimuli reduced the intracellular K+ concentration in Xenopus oocytes and increased caspase-3 activity. The reduction in intracellular K+ was prevented by dense expression of voltage-gated K (Kv) channels. Despite this, the caspase-3 activity was increased similarly in Kv channel-expressing oocytes as in oocytes not expressing Kv channels. Thus, in Xenopus oocytes caspase-3 activity is not dependent on the intracellular concentration of K+.