Gating currents Bezanilla, Francisco
The Journal of general physiology,
07/2018, Letnik:
150, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Many membrane proteins sense the voltage across the membrane where they are inserted, and their function is affected by voltage changes. The voltage sensor consists of charges or dipoles that move in ...response to changes in the electric field, and their movement produces an electric current that has been called gating current. In the case of voltage-gated ion channels, the kinetic and steady-state properties of the gating charges provide information of conformational changes between closed states that are not visible when observing ionic currents only. In this
Milestone, the basic principles of voltage sensing and gating currents are presented, followed by a historical description of the recording of gating currents. The results of gating current recordings are then discussed in the context of structural changes in voltage-dependent membrane proteins and how these studies have provided new insights on gating mechanisms.
Departments of Physiology and Anesthesiology, University of
California at Los Angeles, School of Medicine, Los Angeles,
California
Bezanilla, Francisco
The Voltage Sensor in Voltage-Dependent Ion ...Channels. Physiol. Rev. 80: 555-592, 2000. In voltage-dependent Na, K, or Ca channels, the
probability of opening is modified by the membrane potential. This is
achieved through a voltage sensor that detects the voltage and
transfers its energy to the pore to control its gate. We present here
the theoretical basis of the energy coupling between the electric field
and the voltage, which allows the interpretation of the gating charge
that moves in one channel. Movement of the gating charge constitutes
the gating current. The properties are described, along with
macroscopic data and gating current noise analysis, in relation to the
operation of the voltage sensor and the opening of the channel.
Structural details of the voltage sensor operation were resolved
initially by locating the residues that make up the voltage sensor
using mutagenesis experiments and determining the number of charges per
channel. The changes in conformation are then analyzed based on the
differential exposure of cysteine or histidine-substituted
residues. Site-directed fluorescence labeling is then analyzed as
another powerful indicator of conformational changes that allows time
and voltage correlation of local changes seen by the fluorophores with
the global change seen by the electrophysiology of gating currents and
ionic currents. Finally, we describe the novel results on
lanthanide-based resonance energy transfer that show small distance
changes between residues in the channel molecule. All of the
electrophysiological and the structural information are finally
summarized in a physical model of a voltage-dependent channel in
which a change in membrane potential causes rotation of the S4 segment
that changes the exposure of the basic residues from an internally
connected aqueous crevice at hyperpolarized potentials to an externally
connected aqueous crevice at depolarized potentials.
The Na+/K+-ATPase is a chemical molecular machine responsible for the movement of Na+ and K+ ions across the cell membrane. These ions are moved against their electrochemical gradients, so the ...protein uses the free energy of ATP hydrolysis to transport them. In fact, the Na+/K+-ATPase is the single largest consumer of energy in most cells. In each pump cycle, the protein sequentially exports 3Na+ out of the cell, then imports 2K+ into the cell at an approximate rate of 200 cycles/s. In each half cycle of the transport process, there is a state in which ions are stably trapped within the permeation pathway of the protein by internal and external gates in their closed states. These gates are required to open alternately; otherwise, passive ion diffusion would be a wasteful end of the cell’s energy. Once one of these gates open, ions diffuse from their binding sites to the accessible milieu, which involves moving through part of the electrical field across the membrane. Consequently, ions generate transient electrical currents first discovered more than 30 years ago. They have been studied in a variety of preparations, including native and heterologous expression systems. Here, we review three decades’ worth of work using these transient electrical signals to understand the kinetic transitions of the movement of Na+ and K+ ions through the Na+/K+-ATPase and propose the significance that this work might have to the understanding of the dysfunction of human pump orthologs responsible for some newly discovered neurological pathologies.
Voltage-sensing domains (VSDs) undergo conformational changes in response to the membrane potential and are the critical structural modules responsible for the activation of voltage-gated channels. ...Structural information about the key conformational states underlying voltage activation is currently incomplete. Through the use of experimentally determined residue-residue interactions as structural constraints, we determine and refine a model of the Kv channel VSD in the resting conformation. The resulting structural model is in broad agreement with results that originate from various labs using different techniques, indicating the emergence of a consensus for the structural basis of voltage sensing.
► Resting-state conformation of voltage-sensing domain ► Residue-residue interactions as structural constraints from experiments
How membrane proteins sense voltage Bezanilla, Francisco
Nature reviews. Molecular cell biology,
200804, 2008-Apr, 2008-4-00, 20080401, Letnik:
9, Številka:
4
Journal Article
Recenzirano
The ionic gradients across cell membranes generate a transmembrane voltage that regulates the function of numerous membrane proteins such as ion channels, transporters, pumps and enzymes. The ...mechanisms by which proteins sense voltage is diverse: ion channels have a conserved, positively charged transmembrane region that moves in response to changes in membrane potential, some G-protein coupled receptors possess a specific voltage-sensing motif and some membrane pumps and transporters use the ions that they transport across membranes to sense membrane voltage. Characterizing the general features of voltage sensors might lead to the discovery of further membrane proteins that are voltage regulated.
In voltage-gated potassium channels (VGKC), voltage sensors (VSD) endow voltage-sensitivity to pore domains (PDs) through a not fully understood mechanism. Shaker-like VGKC show domain-swapped ...configuration: VSD of one subunit is covalently connected to its PD by the protein backbone (far connection) and non-covalently to the PD of the next subunit (near connection). VSD-to-PD coupling is not fully explained by far connection only, therefore an additional mechanistic component may be based on near connection. Using tandem dimers of Shaker channels we show functional data distinguishing VSD-to-PD far from near connections. Near connections influence both voltage-dependence of C-type inactivation at the selectivity filter and overall PD open probability. We speculate a conserved residue in S5 (S412 in Shaker), within van der Waals distance from next subunit S4 residues is key for the noncanonical VSD-to-PD coupling. Natural mutations of S412-homologous residues in brain and heart VGKC are related to neurological and cardiac diseases.
Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na(+) channels initiates an action potential, and fast ...inactivation facilitates repolarization of the membrane by the outward K(+) current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.
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.
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.
The ability to modulate cellular electrophysiology is fundamental to the investigation of development, function, and disease. Currently, there is a need for remote, nongenetic, light-induced control ...of cellular activity in two-dimensional (2D) and three-dimensional (3D) platforms. Here, we report a breakthrough hybrid nanomaterial for remote, nongenetic, photothermal stimulation of 2D and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photothermal stimulation at subcellular precision without the need for genetic modification, with laser energies lower than a hundred nanojoules, one to two orders of magnitude lower than Au-, C-, and Si-based nanomaterials. Photothermal stimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves as a powerful toolset for studies of cell signaling within and between tissues and can enable therapeutic interventions.
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