Rikard Blunck

Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement

Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine the extent of this transmembrane movement, we use fluorescent resonance energy transfer between the S4 domain and a reference point in the lipid bilayer. The lipophilic ion dipicrylamine distributes on either side of the lipid bilayer depending on the membrane potential, and is used here as a resonance-energy-transfer acceptor from donor molecules attached to several positions in the Shaker K+ channel. A voltage-driven transmembrane movement of the donor should produce a transient fluorescence change because the acceptor also translocates as a function of voltage. In Shaker K+ channels no such transient fluorescence is observed, indicating that the S4 segment does not translocate across the lipid bilayer. Based on these observations, we propose a molecular model of voltage gating that can account for the observed 13e gating charge with limited transmembrane S4 movement.

A hybrid approach to measuring electrical activity in genetically specified neurons

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.

Bilayer Reconstitution of Voltage-Dependent Ion Channels using a Microfabricated Silicon Chip

Painted bilayers containing reconstituted ion channels serve as a well defined model system for electrophysiological investigations of channel structure and function. Horizontally oriented bilayers with easy solution access to both sides were obtained by painting a phospholipid:decane mixture across a cylindrical pore etched into a 200-microm thick silicon wafer. Silanization of the SiO(2) layer produced a hydrophobic surface that promoted the adhesion of the lipid mixture. Standard lithographic techniques and anisotropic deep-reactive ion etching were used to create pores with diameters from 50 to 200 microm. The cylindrical structure of the pore in the partition and the surface treatment resulted in stable bilayers. These were used to reconstitute Maxi K channels in the 100- and 200-microm diameter pores. The electrophysiological characteristics of bilayers suspended in microchips were comparable with that of other bilayer preparations. The horizontal orientation and good voltage clamping properties make the microchip bilayer method an excellent system to study the electrical properties of reconstituted membrane proteins simultaneously with optical probes.

New Insights Into Endotoxin-Induced Activation of Macrophages: Involvement of a K+ Channel in Transmembrane Signaling

LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators. These regulate the immune response, but may also cause severe harm leading to septic shock. The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14. As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior. Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca2+- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages. The channel is activated by endotoxically active LPS in a dose-dependent manner. Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs. Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K+ channel blockers.

Detection of the Opening of the Bundle Crossing in KcsA with Fluorescence Lifetime Spectroscopy Reveals the Existence of Two Gates for Ion Conduction

The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region. We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.

An intersubunit interaction between S4-S5 linker and S6 is responsible for the slow off-gating component in Shaker K+ channels.

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K+ channels, the gating currents are asymmetric; although the on-gating currents are fast, the off-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between Arg-394, Glu-395, and Leu-398 on the C termini of the S4-S5 linker and Tyr-485 on the S6 of the neighboring subunit, which is responsible for the development of the slow off-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.

Mechanism of Electromechanical Coupling in Voltage-Gated Potassium Channels

Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion – sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3–4e+ each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4–S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during gating.

Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid

Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.

Fluorescence detection of the movement of single KcsA subunits reveals cooperativity

The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step.