Turning a Kv channel into hot and cold receptor by perturbing its electromechanical coupling.

Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a "hot" receptor, reflecting the temperature dependence of the voltage sensor's last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a "cold" receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a "loose" or "tight" conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.

Photolipid excitation triggers depolarizing optocapacitive currents and action potentials.

Optically-induced changes in membrane capacitance may regulate neuronal activity without requiring genetic modifications. Previously, they mainly relied on sudden temperature jumps due to light absorption by membrane-associated nanomaterials or water. Yet, nanomaterial targeting or the required high infrared light intensities obstruct broad applicability. Now, we propose a very versatile approach: photolipids (azobenzene-containing diacylglycerols) mediate light-triggered cellular de- or hyperpolarization. As planar bilayer experiments show, the respective currents emerge from millisecond-timescale changes in bilayer capacitance. UV light changes photolipid conformation, which awards embedding plasma membranes with increased capacitance and evokes depolarizing currents. They open voltage-gated sodium channels in cells, generating action potentials. Blue light reduces the area per photolipid, decreasing membrane capacitance and eliciting hyperpolarization. If present, mechanosensitive channels respond to the increased mechanical membrane tension, generating large depolarizing currents that elicit action potentials. Membrane self-insertion of administered photolipids and focused illumination allows cell excitation with high spatiotemporal control.

Photolipid excitation triggers depolarizing optocapacitive currents and action potentials.

Optically-induced changes in membrane capacitance may regulate neuronal activity without requiring genetic modifications. Previously, they mainly relied on sudden temperature jumps due to light absorption by membrane-associated nanomaterials or water. Yet, nanomaterial targeting or the required high infrared light intensities obstruct broad applicability. Now, we propose a very versatile approach: photolipids (azobenzene-containing diacylglycerols) mediate light-triggered cellular de- or hyperpolarization. As planar bilayer experiments show, the respective currents emerge from millisecond-timescale changes in bilayer capacitance. UV light changes photolipid conformation, which awards embedding plasma membranes with increased capacitance and evokes depolarizing currents. They open voltage-gated sodium channels in cells, generating action potentials. Blue light reduces the area per photolipid, decreasing membrane capacitance and eliciting hyperpolarization. If present, mechanosensitive channels respond to the increased mechanical membrane tension, generating large depolarizing currents that elicit action potentials. Membrane self-insertion of administered photolipids and focused illumination allows cell excitation with high spatiotemporal control.

A mechanistic reinterpretation of fast inactivation in voltage-gated Na+ channels.

The hinged-lid model was long accepted as the canonical model for fast inactivation in Nav channels. It predicts that the hydrophobic IFM motif acts intracellularly as the gating particle that binds and occludes the pore during fast inactivation. However, the observation in recent high-resolution structures that the bound IFM motif is located far from the pore, contradicts this preconception. Here, we provide a mechanistic reinterpretation of fast inactivation based on structural analysis and ionic/gating current measurements. We demonstrate that in Nav1.4 the final inactivation gate is comprised of two hydrophobic rings at the bottom of S6 helices. These rings function in series and close downstream of IFM binding. Reducing the volume of the sidechain in both rings leads to a partially conductive, leaky inactivated state and decreases the selectivity for Na+ ion. Altogether, we present an alternative molecular framework to describe fast inactivation.

A Mechanistic Reinterpretation of Fast Inactivation in Voltage-Gated Na+ Channels.

The hinged-lid model is long accepted as the canonical model for fast inactivation in Nav channels. It predicts that the hydrophobic IFM motif acts intracellularly as the gating particle that binds and occludes the pore during fast inactivation. However, the observation in recent high-resolution structures that the bound IFM motif locates far from the pore, contradicts this preconception. Here, we provide a mechanistic reinterpretation of fast inactivation based on structural analysis and ionic/gating current measurements. We demonstrate that in Nav1.4 the final inactivation gate is comprised of two hydrophobic rings at the bottom of S6 helices. These rings function in series and close downstream of IFM binding. Reducing the volume of the sidechain in both rings leads to a partially conductive "leaky" inactivated state and decreases the selectivity for Na + ion. Altogether, we present an alternative molecular framework to describe fast inactivation.

A Mechanistic Reinterpretation of Fast Inactivation in Voltage-Gated Na + Channels.

Fast Inactivation in voltage-gated Na + channels plays essential roles in numerous physiological functions. The canonical hinged-lid model has long predicted that a hydrophobic motif in the DIII-DIV linker (IFM) acts as the gating particle that occludes the permeation pathway during fast inactivation. However, the fact that the IFM motif is located far from the pore in recent high-resolution structures of Nav + channels contradicts this status quo model. The precise molecular determinants of fast inactivation gate once again, become an open question. Here, we provide a mechanistic reinterpretation of fast inactivation based on ionic and gating current data. In Nav1.4 the actual inactivation gate is comprised of two hydrophobic rings at the bottom of S6. These function in series and closing once the IFM motif binds. Reducing the volume of the sidechain in both rings led to a partially conductive inactivated state. Our experiments also point to a previously overlooked coupling pathway between the bottom of S6 and the selectivity filter.

Noncanonical electromechanical coupling paths in cardiac hERG potassium channel.

Voltage-gated potassium channels are involved in many physiological processes such as nerve impulse transmission, the heartbeat, and muscle contraction. However, for many of them the molecular determinants of the gating mechanism remain elusive. Here, using a combination of theoretical and experimental approaches, we address this problem focusing on the cardiac hERG potassium channel. Network analysis of molecular dynamics trajectories reveals the presence of a kinematic chain of residues that couples the voltage sensor domain to the pore domain and involves the S4/S1 and S1/S5 subunit interfaces. Mutagenesis experiments confirm the role of these residues and interfaces in the activation and inactivation mechanisms. Our findings demonstrate the presence of an electromechanical transduction path crucial for the non-domain-swapped hERG channel gating that resembles the noncanonical path identified in domain-swapped K+ channels.

Ion channel thermodynamics studied with temperature jumps measured at the cell membrane.

Perturbing the temperature of a system modifies its energy landscape, thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage clamp while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 9°C in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with sub-ms time resolution by measuring the time course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir1.1 isoform b, which reveals a highly temperature-sensitive blockage relief, and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane protein thermodynamics.

Optocapacitance: physical basis and its application.

The observation that membrane capacitance increases with temperature has led to the development of new methods of neuronal stimulation using light. The optocapacitive effect refers to a light-induced change in capacitance produced by the heating of the membrane through a photothermal effect. This change in capacitance manifests as a current, named optocapacitive current that depolarizes cells and therefore can be used to stimulate excitable tissues. Here, we discuss how optocapacitance arises from basic membrane properties, the characteristics of the optocapacitive current, its use for neuronal stimulation, and the challenges for its application in vivo.

Metal Bridge in S4 Segment Supports Helix Transition in Shaker Channel.

Voltage-gated ion channels play important roles in physiological processes, especially in excitable cells, in which they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared; the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, the possible helix transition from α- to 310-helix in S4 during the activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, in i, i + 3 and i, i + 6 or i, i + 4 and i, i + 7 pairs, to favor 310- or α-helical conformations, respectively. We have taken advantage of the ability of His to coordinate Zn2+ to promote metal ion bridges, and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in α-helical conformation). In the presence of 20 µM of Zn2+, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, and the voltage dependence is shifted by 10 mV toward depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in α-helical conformation, a closed (resting or intermediate) state is stabilized rather than destabilizing the open (active) state. Taken together, our data indicates that S4 undergoes α-helical conformation to a short-lived different secondary structure transiently before reaching the active state in the activation process.