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Ionic size effects and electrostatic correlations result in the non-monotonic dependence of the electrical conductivity on the pore size. For ion transport at a high gating voltage, the conductivity oscillates with the pore size due to a significant overlap of the electric double layers.
Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. In this report we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNa V ) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins.
With the ability to selectively control ionic flux, biological protein ion channels perform a fundamental role in many physiological processes. For practical applications that require the functionality of a biological ion channel, graphene provides a promising solid-state alternative, due to its atomic thinness and mechanical strength. Here, we demonstrate that nanopores introduced into graphene membranes, as large as 50 nm in diameter, exhibit inter-cation selectivity with a ~20× preference for K + over divalent cations and can be modulated by an applied gate voltage. Liquid atomic force microscopy of the graphene devices reveals surface nanobubbles near the pore to be responsible for the observed selective behavior. Molecular dynamics simulations indicate that translocation of ions across the pore likely occurs via a thin water layer at the edge of the pore and the nanobubble. Our results reflect a significant improvement in the inter-cation selectivity displayed by a solid-state nanopore device and by utilizing the pores in a de-wetted state, offers a method to fabricate selective graphene membranes that does not rely on the fabrication of sub-nm pores.
Over two-thirds of a century ago, Hodgkin and Huxley proposed the existence of voltage gated ion channels (VGICs) to carry Na + and K + ions across the cell membrane to create the nerve impulse, in response to depolarization of the membrane. The channels have multiple physiological roles, and play a central role in a wide variety of diseases when they malfunction. The first channel structure was found by MacKinnon and coworkers in 1998. Subsequently, the structure of a number of VGICs was determined in the open (ion conducting) state. This type of channel consists of four voltage sensing domains (VSDs), each formed from four transmembrane (TM) segments, plus a pore domain through which ions move. Understanding the gating mechanism (how the channel opens and closes) requires structures. One TM segment (S4) has an arginine in every third position, with one such segment per domain. It is usually assumed that these arginines are all ionized, and in the resting state are held toward the intracellular side of the membrane by voltage across the membrane. They are assumed to move outward (extracellular direction) when released by depolarization of this voltage, producing a capacitive gating current and opening the channel. We suggest alternate interpretations of the evidence that led to these models. Measured gating current is the total charge displacement of all atoms in the VSD; we propose that the prime, but not sole, contributor is proton motion, not displacement of the charges on the arginines of S4. It is known that the VSD can conduct protons. Quantum calculations on the K v 1.2 potassium channel VSD show how; the key is the amphoteric nature of the arginine side chain, which allows it to transfer a proton. This appears to be the first time the arginine side chain has had its amphoteric character considered. We have calculated one such proton transfer in detail: this proton starts from a tyrosine that can ionize, transferring to the NE of the third arginine on S4; that arginine’s NH then transfers a proton to a glutamate. The backbone remains static. A mutation predicted to affect the proton transfer has been qualitatively confirmed experimentally, from the change in the gating current-voltage curve. The total charge displacement in going from a normal closed potential of -70 mV across the membrane to 0 mV (open), is calculated to be approximately consistent with measured values, although the error limits on the calculation require caution in interpretation.
Ion channels are ubiquitous throughout all forms of life. Potassium channels are even found in viruses. Every cell must communicate with its surroundings, so all cells have them, and excitable cells, in particular, especially nerve cells, depend on the behavior of these channels. Every channel must be open at the appropriate time, and only then, so that each channel opens in response to the stimulus that tells that channel to open. One set of channels, including those in nerve cells, responds to voltage. There is a standard model for the gating of these channels that has a section of the protein moving in response to the voltage. However, there is evidence that protons are moving, rather than protein. Water is critical as part of the gating process, although it is hard to see how this works in the standard model. Here, we review the extensive evidence of the importance of the role of water and protons in gating these channels. Our principal example, but by no means the only example, will be the Kv1.2 channel. Evidence comes from the effects of D2O, from mutations in the voltage sensing domain, as well as in the linker between that domain and the gate, and at the gate itself. There is additional evidence from computations, especially quantum calculations. Structural evidence comes from X-ray studies. The hydration of ions is critical in the transfer of ions in constricted spaces, such as the gate region and the pore of a channel; we will see how the structure of the hydrated ion fits with the structure of the channel. In addition, there is macroscopic evidence from osmotic experiments and streaming current measurements. The combined evidence is discussed in the context of a model that emphasizes the role of protons and water in gating these channels.
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Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa V ) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa V CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa V CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Altogether, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa V voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.
The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD). The primary supporting evidence came from R→C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS) reagent. The cys side chain is –SH (reactive form –S−); the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain –OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.
CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl – -permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl – -permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl – -permeation pathway.
Voltage-gated sodium channels, Na V s, are responsible for the rapid rise of action potentials in excitable tissues. Na V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na V 1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na V isoform has been challenging because they usually show undesired crossreactivity for different Na V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa V 1.4 or CTNa V 1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa V 1.4 and CTNa V 1.5 with high affinity (K D ~ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na V 1.4 and Na V 1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na V 1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na V reagents to capture Na V s from cell lysates and as molecular visualization agents for Na V s.
The separation of rare earth elements (REEs) has historically been a complicated and expensive process, producing significant amounts of pollution. To this end, MXene membranes (MXMs) have attracted researchers' interest. It has been demonstrated that MXMs are capable of separating ions and that it is possible to precisely control the separation rate by applying a voltage to the membrane. Indeed, due to their chemical nature, MXenes show outstanding electrical conductivity. However, the effect of applied voltage on the permeation rate and separation effects of iconic species remains an intriguing area of research. In this work, we demonstrate how applying different voltages (gate voltage) to a MXM affects the permeation rate of various REE ions. Our hypothesis to explain MXMs behavior towards REE is that under negative voltage, the interlayer spacing decreases due to attraction between charged MXene surfaces and intercalated cations, increasing the separation factor between REEs. Under positive voltage, the interlayer spacing increases due to repulsion between the MXene surfaces and intercalated cations. However, we demonstrate relatively poor separation capability for REEs at 1mM starting concentration and 1.0 V applied DC voltage (separation factor ~1-3). This study demonstrates the need for future experiments to observe varied experimental conditions and chemical functionalization to determine whether MXene membranes can be an efficient platform for REE separation.
We present a proposal to realize a perfect electrical switching of topological edge-state transport in a HgTe quantum well (QW). In our device design, we place a strip-like top gate voltage in a conventional quantum-point-contact (QPC) region in the HgTe QW. The numerical calculations show that upon increasing the gate voltage, two new conductance channels are developed in the transport direction and just neighbouring the boundaries of the top gate. The quantum states in the new channels can couple with the edge states to open a gap in energy spectrum, and in turn the gap width can be adjusted by the gate voltage, indicating that switch-on/off of the edge channels can be manipulated in a controllable way. Our device can not only be considered as a development of the conventional QPC structure based on the HgTe QW but also provides a new route to realize topological electrical switchers.
Excitation–contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (Ca V 1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different Ca V isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. Here, the interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II–III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of Ca V 1.2.
Two-dimensional (2D) materials have recently attracted wide attention and rapidly established themselves in various applications. In particular, 2D materials are regarded as promising building blocks for gas sensors due to their high surface-to-volume ratio, ease in miniaturization, and flexibility in enabling wearable electronics. Compared with other 2D materials, MoS{sub 2} is particularly intriguing because it has been widely researched and exhibits semiconducting behavior. Here, we have fabricated MoS{sub 2} resistor based O{sub 2} sensors with a back gate configuration on a 285 nm SiO{sub 2}/Si substrate. The effects of applying back gate voltage stress on O{sub 2} sensing performance have been systematically investigated. With a positive gate voltage stress, the sensor response improves and the response is improved to 29.2% at O{sub 2} partial pressure of 9.9 × 10{sup −5} millibars with a +40 V back-gate bias compared to 21.2% at O{sub 2} partial pressure of 1.4 × 10{sup −4} millibars without back-gate bias; while under a negative gate voltage stress of −40 V, a fast and full recovery can be achieved at room temperature. In addition, a method in determining O{sub 2} partial pressure with a detectability as low as 6.7 × 10{sup −7} millibars at a constant vacuum pressure is presented and its potential as a vacuum gauge is briefly discussed.
The mobility of emerging (e.g., two-dimensional, oxide, organic) semiconductors is commonly estimated from transistor current-voltage measurements. However, such devices often experience contact gating, i.e., electric fields from the gate modulate the contact resistance during measurements, which can lead conventional extraction techniques to estimate mobility incorrectly even by a factor >2. Although this error can be minimized by measuring transistors at high gate-source bias |V gs |, this regime is often inaccessible in emerging devices that suffer from high contact resistance or early gate dielectric breakdown. Here, we propose a method of extracting mobility in transistors with gate-dependent contact resistance that does not require operation at high |V gs |, enabling accurate mobility extraction even in emerging transistors with strong contact gating. Our approach relies on updating the transfer length method (TLM) and can achieve <10% error even in regimes where conventional techniques overestimate mobility by >2×
Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca 2+ . Ca 2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca 2+ or Ba 2+ can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Here, our Ba 2+ -bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels.