Modeling of mechanosensitive channel gating in response to wall shear stress

The accurate biological function of mechanosensitive (MS) channels is crucial for maintaining the viability of living cells. For instance, in vascular endothelial cells, calcium influx from the extracellular environment into cytoplasm is regulated by stretch-activated channels. However, the mechanism by which cells sense force remains unclear. For this study, we hypothesized that gating of ion channels is simply regulated by the direct mechanical stress induced in a membrane. We modeled a membrane channel using crystallographic data of the bacteria Mycobacterium tuberculosis (Tb-MscL) because MscL homologs are integral membrane proteins with sequence similarity to most known ion channels. Molecular dynamics (MD) simulations were performed to elucidate the gating mechanism of the channel protein in response to the fluid shear stress. Results suggest that the stretched membrane drives the interfacial part of the protein–membrane complex to expand and maintains the stability of the constricted part of the transmembrane pore. Moreover, structural similarities between Tb-MscL and the family of ligand-gated ion channels suggest that the conformational change of this model in response to fluid shear stress is useful for modeling the gating mechanism in a broad class of gated channels.     

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K⁺ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.     

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.     

Kinetics of light-sensitive channels in vertebrate photoreceptors

We have studied the ion channels mediating the light response of vertebrate rod photoreceptors by analysing fluctuations in the current across the rod membrane, using the whole cell patch-clamp technique on rods isolated from the axolotl retina. Light decreases the membrane current fluctuations. Noise analysis reveals two components to this decrease: a low frequency component due to biochemical noise in the transduction mechanism, and a high frequency component we attribute to the random opening and closing of the ion channels in the dark. The probability of any one channel being open in the dark is low. The spectrum of the high frequency component of the current fluctuations indicates that the current through an open channel is 4 X 10(-15)A, that the mean channel open time is 2 ms, and that about 10000 channels are open in each rod in the dark. The effect of light is to reduce the opening rate constant of these channels, with no effect on the closing rate constant.     

Calmodulin contributes to gating control in olfactory calcium-activated chloride channels

In sensory neurons of the peripheral nervous system, receptor potentials can be amplified by depolarizing Cl currents. In mammalian olfactory sensory neurons (OSNs), this anion-based signal amplification results from the sequential activation of two distinct types of transduction channels: cAMP-gated Ca channels and Ca-activated Cl channels. The Cl current increases the initial receptor current about 10-fold and leads to the excitation of the neuron. Here we examine the activation mechanism of the Ca-dependent Cl channel. We focus on calmodulin, which is known to mediate Ca effects on various ion channels. We show that the cell line Odora, which is derived from OSN precursor cells in the rat olfactory epithelium, expresses Ca-activated Cl channels. Single-channel conductance, ion selectivity, voltage dependence, sensitivity to niflumic acid, and Ca sensitivity match between Odora channels and OSN channels. Transfection of Odora cells with CaM mutants reduces the Ca sensitivity of the Cl channels. This result points to the participation of calmodulin in the gating process of Ca-ativated Cl channels, and helps to understand how signal amplification works in the olfactory sensory cilia. Calmodulin was previously shown to mediate feedback inhibition of cAMP-synthesis and of the cAMP-gated Ca channels in OSNs. Our results suggest that calmodulin may also be instrumental in the generation of the excitatory Cl current. It appears to play a pivotal role in the peripheral signal processing of olfactory sensory information. Moreover, recent results from other peripheral neurons, as well as from smooth muscle cells, indicate that the calmodulin-controlled, anion-based signal amplification operates in various cell types where it converts Ca signals into membrane depolarization.     

Functional properties of threefold and fourfold channels in ferritin deduced from electrostatic calculations

The iron storage protein ferritin contains threefold and fourfold symmetric channels that are thought to provide pathways for the transfer of Fe(2+) ions in and out of the protein. Using the known crystal structure of the ferritin protein, we perform electrostatic potential energy calculations to elucidate the functional properties of these channels. The threefold channel is shown to be responsible for the transit of Fe(2+) ions. Monovalent ions can also diffuse through the threefold channel but presence of divalent ions in the pore retards this process leading to a selectivity mechanism similar to the one observed in calcium channels. The fourfold channel is found to be impermeant to all cations with the possible exception of protons. Because proton transfer is essential to maintain the electroneutrality of the protein during iron deposition, we suggest that the function of the fourfold channel is to form a "proton wire" that facilitates their transfer in and out of ferritin.     

Bi-stability, hysteresis, and memory of voltage-gated lysenin channels

Lysenin, a 297 amino acid pore-forming protein extracted from the coelomic fluid of the earthworm E. foetida, inserts constitutively open large conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels show voltage regulation and slowly close at positive applied voltages. We report on the consequences of slow voltage-induced gating of lysenin channels inserted into a planar Bilayer Lipid Membrane (BLM), and demonstrate that these pore-forming proteins constitute memory elements that manifest gating bi-stability in response to variable external voltages. The hysteresis in macroscopic currents dynamically changes when the time scale of the voltage variation is smaller or comparable to the characteristic conformational equilibration time, and unexpectedly persists for extremely slow-changing external voltage stimuli. The assay performed on a single lysenin channel reveals that hysteresis is a fundamental feature of the individual channel unit and an intrinsic component of the gating mechanism. The investigation conducted at different temperatures reveals a thermally stable reopening process, suggesting that major changes in the energy landscape and kinetics diagram accompany the conformational transitions of the channels. Our work offers new insights on the dynamics of pore-forming proteins and provides an understanding of how channel proteins may form an immediate record of the molecular history which then determines their future response to various stimuli. Such new functionalities may uncover a link between molecular events and macroscopic processing and transmission of information in cells, and may lead to applications such as high density biologically-compatible memories and learning networks.     

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 channel's 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.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.     

The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes

The influence of static magnetic fields (SMFs) on the activity of recombinant mechanosensitive ion channels (the bacterial mechanosensitive ion channel of large conductance—MscL) following reconstitution into artificial liposomes has been investigated. Preliminary findings suggest that exposure to 80-mT SMFs does not induce spontaneous MscL activation in the absence of mechanical stimulation. However, SMFs do appear to influence the open probability and single channel kinetics of MscL exposed to negative pipette pressure. Typical responses include an overall reduction in channel activity or an increased likelihood of channels becoming trapped open in sub-conducting states following exposure to SMFs. There is a delay in the onset of this effect and it is maintained throughout exposure. Generally, channel activity showed slow or limited recovery following removal of the magnetic field and responses to the magnetic were often reduced or abolished upon subsequent exposures. Pre-exposure of the liposomes to SMFs resulted in reduced sensitivity of MscL to negative pipette pressure, with higher pressures required to activate the channels. Although the mechanisms of this effect are not clear, our initial observations appear to support previous work showing that the effects of SMFs on ion channels may be mediated by changes in membrane properties due to anisotropic diamagnetism of lipid molecules.     

N-glycosylation of TRPM8 ion channels modulates temperature sensitivity of cold thermoreceptor neurons

TRPM8 is a member of the transient receptor potential ion channel superfamily, which is expressed in sensory neurons and is activated by cold and cooling compounds, such as menthol. Activation of TRPM8 by agonists takes place through shifts in its voltage activation curve, allowing channel opening at physiological membrane potentials. Here, we studied the role of the N-glycosylation occurring at the pore loop of TRPM8 on the function of the channel. Using heterologous expression of recombinant channels in HEK293 cells we found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild type channel. These differences include a shift in the threshold of temperature activation and a reduced response to menthol and cold stimuli. Biophysical analysis indicated that these modifications are due to a shift in the voltage dependence of TRPM8 activation toward more positive potentials. By using tunicamycin, a drug that prevents N-glycosylation of proteins, we also evaluated the effect of the N-glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. These experiments showed that the lack of N-glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results indicate that post-translational modification of TRPM8 is an important mechanism modulating cold thermoreceptor function, explaining the marked differences in temperature sensitivity observed between recombinant and native TRPM8 ion channels.     

Extremely low frequency magnetic fields can either increase or decrease analgaesia in the land snail depending on field and light conditions

Results of prior investigations with opioid peptide mediated antinociception or analgaesia have suggested that these extremely low frequency (ELF) magnetic field effects are described by a resonance mechanism rather than mechanisms based on either induced currents or magnetite. Here we show that ELF magnetic fields (141-414 microT peak) can, in a manner consistent with the predictions of Lednev's parametric resonance model (PRM) for the calcium ion, either (i) reduce, (ii) have no effect on, or (iii) increase endogenous opioid mediated analgaesia in the land snail, Cepaea nemoralis. When the magnetic fields were set to parameters for the predictions of the PRM for the potassium ion, opioid-peptide mediated analgaesia increased and there was evidence of antagonism by the K(+) channel blocker, glibenclamide. Furthermore, these effects were dependent on the presence of light; the effects were absent in the absence of light. These observed increases and decreases in opioid analgaesia are largely consistent with the predictions of Lednev's PRM.     

Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in cytosolic C tail of Slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels

Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Application of oscillating potentials to the Shaker potassium channel

Nonequilibrium response spectroscopy (NRS) has been proposed recently to complement standard electrophysiological techniques used to investigate ion channels. It involves application of rapidly oscillating potentials that drive the ion channel ensemble far from equilibrium. It is argued that new, so far undiscovered features of ion channel gating kinetics may become apparent under such nonequilibrium conditions. In this paper we explore the possibility of using regular, sinusoidal voltages with the NRS protocols to facilitate Markov model selection for ion channels. As a test case we consider the Shaker potassium channel for which various Markov models have been proposed recently. We concentrate on certain classes of such models and show that while some models might be virtually indistinguishable using standard methods, they show marked differences when driven with an oscillating voltage. Model currents are compared to experimental data obtained for the Shaker K+ channel expressed in mammalian cells (tsA 201).     

High spatial density is associated with non-conducting Kv channels from two families

Ion channels are well known for their ability to regulate the cell membrane potential. However, many ion channels also have functions that do not involve ion conductance. Kv2 channels are one family of ion channels whose non-conducting functions are central to mammalian cell physiology. Kv2.1 and Kv2.2 channels form stable contact sites between the endoplasmic reticulum and plasma membrane via an interaction with endoplasmic reticulum resident proteins. To perform this structural role, Kv2 channels are expressed at extremely high densities on the plasma membranes of many cell types, including central pyramidal neurons, α-motoneurons, and smooth muscle cells. Research from our lab and others has shown that the majority of these plasma membrane Kv2.1 channels do not conduct potassium in response to depolarization. The mechanism of this channel silencing is unknown but is thought to be dependent on channel density in the membrane. Furthermore, the prevalence of a non-conducting population of Kv2.2 channels has not been directly tested. In this work we make improved measurements of the numbers of conducting and non-conducting Kv2.1 channels expressed in HEK293 cells and expand the investigation of non-conducting channels to three additional Kv α-subunits: Kv2.2, Kv1.4, and Kv1.5. By comparing the numbers of gating and conducting channels in individual HEK293 cells, we found that on average, only 50% of both Kv2.1 and Kv2.2 channels conducted potassium and, as previously suggested, that fraction decreased with increased channel density in the plasma membrane. At the highest spatial densities tested, which are comparable with those found at Kv2 clusters in situ, only 20% of Kv2.1 and Kv2.2 channels conducted potassium. We also show for the first time that Kv1.4 and Kv1.5 exhibit density-dependent silencing, suggesting that this phenomenon has an underlying mechanism that is shared by Kv channels from multiple families.     

Sigma receptor photolabeling and sigma receptor-mediated modulation of potassium channels in tumor cells.

Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membrane-spanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and (+/-)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (IK). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition in IK, and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 microM. SKF10047 also inhibited IK with a similar EC50 (14 microM). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5'-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.     

Haloperidol induces calcium ion influx via L-type calcium channels in hippocampal HN33 cells and renders the neurons more susceptible to oxidative stress

Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of pro-apoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate.