Endogenous Ion Channels Research Articles

Synthetic Peptide-Based Channels: Candidates for Treatments of Channelopathies

Modern approaches for treatments of channelopathies employ mutation-specific pharmacotherapies, use pharmacoagents alone, or a vast variety of ideas for genetic approaches, all with the purpose of restoring defective ion channels or attenuate their negative effects. An alternative to existing approaches is the use of synthetic channel-forming peptides (CFPs) with desirable selectivity, high ion transport rates and overall ability to supersede defective endogenous ion channels. Our synthetic CFPs represent derivatives of a peptide initially reconstituted from the second transmembrane segment of the α-subunit of Glycine receptor (M2GlyR), PARVGLGITTVLTMTTQSSGSRA.

Characterization of Eag1 Channel Lateral Mobility in Rat Hippocampal Cultures by Single-Particle-Tracking with Quantum Dots

Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been achieved yet. Using a specific extracellular antibody and quantum dots we reveal and characterize lateral mobility as a faster mechanism to dynamically control the number of endogenous ether-a-go-go (Eag)1 ion channels inside synapses. We visualize Eag1 entering and leaving synapses by lateral diffusion in the plasma membrane of rat hippocampal neurons. Mathematical analysis of their trajectories revealed how the motion of Eag1 gets restricted when the channels diffuse into the synapse, suggesting molecular interactions between Eag1 and synaptic components. In contrast, Eag1 channels switch to Brownian movement when they exit synapses and diffuse into extrasynaptic membranes. Furthermore, we demonstrate that the mobility of Eag1 channels is specifically regulated inside synapses by actin filaments, microtubules and electrical activity. In summary, using single-particle-tracking techniques with quantum dots nanocrystals, our study shows for the first time the lateral diffusion of an endogenous voltage-gated ion channel in neurons. The location-dependent constraints imposed by cytoskeletal elements together with the regulatory role of electrical activity strongly suggest a pivotal role for the mobility of voltage-gated ion channels in synaptic activity.

Control of embryonic stem cell proliferation and migration can be controlled in vivo by pharmacological modulation of endogenous ion channels

Control of embryonic stem cell proliferation and migration can be controlled in vivo by pharmacological modulation of endogenous ion channels

The Role of Aβ-Induced Calcium Dysregulation in the Pathogenesis of Alzheimer's Disease

Although many of the biochemical mechanisms which regulate production or clearance of the amyloid-beta protein (Abeta) of Alzheimer's disease (AD) are now well understood, the mechanism of Abeta neurotoxicity remains unclear. A number of studies have shown that Abeta can disrupt neuronal Ca(2+) homeostasis by inducing influx of extracellular Ca(2+) into the neuronal cytoplasm. Ca(2+) is known to play an important role in neuronal excitability, synaptic plasticity and neurotoxicity. Therefore, Abeta-induced Ca(2+) dysregulation may contribute to many of the cognitive and neuropathologic features of AD. In vitro studies show that Abeta can increase ion permeability in lipid membranes. This increased permeability is reportedly associated with the formation of artificial ion pores formed from Abeta oligomers. However, a number of other studies show that Abeta can activate endogenous ion channels on the cell surface. There is also increasing evidence that presenilin mutations alter intracellular Ca(2+) stores. It is likely that elucidation of the mechanism by which Abeta and presenilin cause Ca(2+) dysregulation in neurons will help to identify new drug targets for the treatment of AD.

Photochemical control of endogenous ion channels and cellular excitability

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Alterations in intracellular potassium concentration by HIV-1 and SIV Nef

BackgroundHIV-1 mediated perturbation of the plasma membrane can produce an alteration in the transmembrane gradients of cations and other small molecules leading to cell death. Several HIV-1 proteins have been shown to perturb membrane permeability and ion transport. Xenopus laevis oocytes have few functional endogenous ion channels, and have proven useful as a system to examine direct effects of exogenously added proteins on ion transport.ResultsHIV-1 Nef induces alterations in the intracellular potassium concentration in CD4+ T-lymphoblastoid cells, but not intracellular pH. Two electrode voltage-clamp recording was used to determine that Nef did not form ion channel-like pores in Xenopus oocytes.ConclusionThese results suggest that HIV-1 Nef regulates intracellular ion concentrations indirectly, and may interact with membrane proteins such as ion channels to modify their electrical properties.

P2Y1 receptor modulation of endogenous ion channel function in Xenopus oocytes: Involvement of transmembrane domains

Agonist activation of the hP2Y1 receptor expressed in Xenopus oocytes stimulated an endogenous voltage-gated ion channel, previously identified as the transient inward (Tin) channel. When human P2Y1 (hP2Y1) and skate P2Y (sP2Y) receptors were expressed in Xenopus oocytes, time-to-peak values (a measure of the response to membrane hyperpolarization) of the Tin channel were significantly reduced compared to oocytes expressing the hB1-bradykinin receptor or the rat M1-muscarinic (rM1) receptor. Differences in activation were also observed in the Tin currents elicited by various P2Y receptor subtypes. The time-to-peak values of the Tin channel in oocytes expressing the hP2Y4, hP2Y11, or hB1-bradykinin receptors were similar, whereas the channel had significantly shorter time-to-peak values in oocytes expressing either the hP2Y1 or sP2Y receptor. Amino acid substitutions at His-132, located in the third transmembrane domain (TM3) of the hP2Y1 receptor, delayed the onset of channel opening, but not the kinetics of the activation process. In addition, Zn2+ sensitivity was also dependent on the subtype of P2Y receptor expressed. Replacement of His-132 in the hP2Y1 receptor with either Ala or Phe increased Zn2+ sensitivity of the Tin current. In contrast, truncation of the C-terminal region of the hP2Y1 receptor had no affect on activation or Zn2+ sensitivity of the Tin channel. These results suggested that TM3 in the hP2Y1 receptor was involved in modulating ion channel function and blocker pharmacology of the Tin channel.

P2Y Receptors Modulate Ion Channel Function through Interactions Involving The C-Terminal Domain

Nucleotide stimulation of G(q)-coupled P2Y receptors expressed in Xenopus laevis oocytes produces the activation of an endogenous voltage-gated ion channel, previously identified as the transient inward (T(in)) channel. Expression of human P2Y(1), human P2Y(2), rat P2Y(6), human P2Y(11), or skate P2Y receptors in oocytes resulted in modulation of the voltage dependence and inactivation gating of the channel. Expression of the human P2Y(4) receptor, rat M(1)-muscarinic receptor, and human B(1)-bradykinin receptor did not alter the properties of the T(in) channel. Replacement of the C-terminal domain of the human B(1)-bradykinin receptor with the C-terminal domains of either the human P2Y(1) or human P2Y(2) receptor resulted in voltage dependence and inactivation-gating properties, respectively, of the T(in) channel that were similar to those elicited by the respective native P2Y receptor. Systematic truncation of the C-terminal region of the human P2Y(1) receptor identified a short region responsible for modulation of the T(in) channel. This region contains a conserved sequence motif found in all P2Y receptors that modulates the voltage dependence of the T(in) channel. Synthetic 20-mer peptides from the C-terminal domains of human P2Y(1) and P2Y(2) receptors produced a shift in the voltage dependence and slowed inactivation gating, respectively, after injection into oocytes expressing human B(1)-bradykinin or truncated human P2Y(1) receptors. These results indicate that certain P2Y receptors are capable of modulating the voltage sensitivity and inactivation gating of an endogenous oocyte ion channel through interactions involving the C-terminal region of the receptor. Such modulation of ion channel function could also exist in native mammalian cells that express P2Y receptors.

Low pH modulation of recombinant vanilloid receptors and perivascular capsaicin-sensitive sensory neurotransmission

The effect of low pH on capsaicin-sensitive sensory neurotransmission in the rat isolated mesenteric arterial bed and at recombinant (rVR1) vanilloid receptors was investigated. Mesenteric sensory neurogenic vasorelaxation elicited by electrical field stimulation was reversibly inhibited by lowering pH from 7.4 to 6.9 and 6.3. Capsaicin-induced vasorelaxation was not different at pH 6.9, but was attenuated at pH 6.3. Vasorelaxation to calcitonin gene-related peptide, the principal sensory motor neurotransmitter in rat mesenteric arteries, was not different at pH 6.9 or pH 6.3. In rVR1-transfected HEK293 cells, acidic conditions enhanced the affinities of capsaicin and capsazepine at rVR1, but did not affect the potency of carbachol at endogenous muscarinic receptors. Following inactivation of endogenous acid-sensitive ion channels, lowering pH (6.0–4.5) directly increased [Ca 2+] i in rVR1-HEK293 cells (EC 50 5.5). This response was abolished by 1 μM capsazepine. In conclusion, a decrease in pH (to 6.9 and 6.3) enhances the affinity of capsaicin at rVR1, but inhibits sensory neurotransmission in the rat mesenteric arterial bed. This likely explains why there is no evidence of an enhancement of sensitivity to capsaicin at endogenous vanilloid receptors, as observed with rVR1. When pH is reduced still further (6.0–5.5) there is direct activation of rVR1.

GOLAC: An Endogenous Anion Channel of the Golgi Complex

The Golgi complex is present in every eukaryotic cell and functions in posttranslational modifications and sorting of proteins and lipids to post-Golgi destinations. Both functions require an acidic lumenal pH and transport of substrates into and by-products out of the Golgi lumen. Endogenous ion channels are expected to be important for these features, but none has been described. Ion channels from an enriched Golgi fraction cleared of transiting proteins were incorporated into planar lipid bilayers. Eighty percent of the single-channel recordings revealed the same anion channel. This channel has novel properties and has been named GOLAC (Golgi anion channel). The channel has six subconductance states with a maximum conductance of 130 pS, is open over 95% of the time, and is not voltage-gated. Significant for Golgi function, the channel conductance is increased by reduction of pH on the lumenal surface. This channel may serve two nonexclusive functions: providing counterions for the acidification of the Golgi lumen by the H +-ATPase and removal of inorganic phosphate generated by glycosylation and sulfation of proteins and lipids in the Golgi.

Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2.

We evaluated the suitability of the S2 foetal Drosophila cell line as an expression system for vertebrate anion channel proteins (e.g. cystic fibrosis transmembrane conductance regulator, CFTR) in patch-clamp studies of the endogenous ion channels. In the inside-out configuration (symmetric 150 mM Cl-) we found most frequently an inwardly rectifying Cl- channel with single-channel conductances (gamma) of 57, 45 and 17 pS at -80, 0 and 80 mV, respectively. Reduction of bath [Cl-] to 40 mM caused a shift in reversal potential (Vrev) to -22.5 mV indicating pronounced Cl- selectivity. In the outside-out configuration ([Cl-]pipette = 40 mM, [Cl-]bath = 150 mM) we observed a Cl- channel with a linear unitary current/voltage (i/V) relation for which gamma was 30 pS. The kinetics were quite slow in both configurations. Cl-selectivity was also observed in whole-cell experiments ([Cl-]pipette = 40 mM) in which a Vrev of -43.8 mV, i.e. close to the Cl- equilibrium potential, demonstrated that the membrane current was dominated by Cl-. We conclude that the important features making S2 cells suitable as an expression system for heterologous expressed anion channel proteins are: small total whole-cell currents (less than 100 pA), single-channel and whole-cell currents that, unlike those of CFTR, cannot be described by the Goldman-Hodgkin-Katz regime, and slow kinetics distinctly different from those of CFTR.

Membrane interaction of TNF is not sufficient to trigger increase in membrane conductance in mammalian cells

Tumor necrosis factor (TNF) can trigger increases in membrane conductance of mammalian cells in a receptor-independent manner via its lectin-like domain. A lectin-deficient TNF mutant, lacking this activity, was able to bind to artificial liposomes in a pH-dependent manner, but not to insert into the bilayer, just like wild type TNF. A peptide mimicking the lectin-like domain, which can still trigger increases in membrane currents in cells, failed to interact with liposomes. Thus, the capacity of TNF to trigger increases in membrane conductance in mammalian cells does not correlate with its ability to interact with membranes, suggesting that the cytokine does not form channels itself, but rather interacts with endogenous ion channels or with plasma membrane proteins that are coupled to ion channels.

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, I C, was activated by perfusing naı̈ve oocytes with a divalent-free recording solution. Ethanol (25–100 mM) modestly inhibited I C with 100 mM ethanol producing a 7–8% inhibition of steady state currents. The store-operated or capacitative calcium current (I SOC) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10–100 mM) dose-dependently inhibited the calcium-dependent chloride current (I Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I Cl(Ca), I Cl-1 and I Cl-2. Under these conditions, ethanol (100 mM) inhibited I Cl-1 currents to a greater extent (38%) than it did I Cl-2 currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.

The lectin-like domain of tumor necrosis factor-alpha increases membrane conductance in microvascular endothelial cells and peritoneal macrophages.

Herein, we show that TNF exerts a pH-dependent increase in membrane conductance in primary lung microvascular endothelial cells and peritoneal macrophages. This effect was TNF receptor-independent, since it also occurred in cells isolated from mice deficient in both types of TNF receptors. A TNF mutant in which the three amino acids critical for the lectin-like activity were replaced by an alanine did not show any significant effect on membrane conductance. Moreover, a synthetic 17-amino acid peptide of TNF, which was previously shown to exert lectin-like activity, also increased the ion permeability in these cells. The amiloride sensitivity of the observed activity suggests a binding of TNF to an endogenous ion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF-mediated vascular pathology.

Endogenous ion channels in oocytes of xenopus laevis: recent developments.

Endogenous ion channels in oocytes of xenopus laevis: recent developments.

Near-visible ultraviolet light induces a novel ubiquitous calcium-permeable cation current in mammalian cell lines.

1. We studied the immediate and short-term effects of UV light in the near-visible range at the cellular and membrane level using the whole-cell patch-clamp technique in combination with digital fluorescence imaging. 2. Illumination with monochromatic UVA light (340-380 nm) induced a sustained non-saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells. 3. The current was non-selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light-induced non-selective cation current. 4. A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tertbutylhydroperoxide, but not by cytosolic oxidized glutathione. 5. The free-radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect. 6. The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell-free membrane patches showing a similar wavelength dependence. 7. These results suggest that ILiNC is activated by UVA light-induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical-induced membrane damage.

A novel explanation for fluctuations of ion current through narrow pores.

Fluctuation of ion current, between a high conductance and a low conductance state, through biological ion channels and pores is assumed to arise from conformational changes between an "open" and a "closed" configuration. Here we offer an additional mechanism that arises from changes in ionization of fixed charges within, or at the mouth of, a channel or pore. Our hypothesis, which is based on measurements of ion selectivity alongside ion current, applies to pores through some synthetic membranes and through channels-such as those created by certain toxins-that remain (at least partially) open in the low conductance state. It may also explain the phenomena of "open channel noise" and "substate behavior" that characterize several endogenous ion channels and should be considered when modeling the behavior of such channels.

Mu-opioid receptor regulates CFTR coexpressed in Xenopus oocytes in a cAMP independent manner.

The objective of this study was to characterize the signaling mechanisms of the mu-opioid receptor in its coupling to the cystic fibrosis transmembrane conductance regulator (CFTR) when coexpressed in Xenopus oocytes. Because oocytes do not contain endogenous cAMP-regulated ion channels, the cAMP-modulated CFTR was coexpressed with receptors as a 'reporter' channel. Agonist treatment of oocytes coexpressing mu-opioid receptors, beta2-adrenergic receptors and CFTR produced Cl- currents in a dose-related manner and immunocytochemical analysis confirmed receptor expression. These data suggest that opioid agonists could activate adenylyl cyclase in this system to elevate cAMP levels. Heterotrimeric G protein betagamma-subunits acting on adenylyl cyclase type II would increase cAMP levels. The probable presence of adenylyl cyclase type II and other components of opioid signal transduction such as G(i alpha2), were demonstrated by RT-PCR. However, measurement of cAMP levels in individual oocytes by radioimmunoassay showed that opioid agonist application to oocytes expressing mu-opioid receptors, beta2-adrenergic receptors and CFTR did not increase cAMP levels, whereas application of the beta2-adrenergic agonist, isoproterenol, or IBMX alone did increase cAMP levels. Opioid-induced CFTR activation was not affected by either application of the broad spectrum kinase inhibitor, H7, nor by application of the specific PKA inhibitor, KT5720. Injection of free betagamma-subunits, which could activate the endogenous type II cyclase, was unable to produce measurable currents in oocytes expressing the CFTR. These studies indicate that opioid activation of the CFTR is not mediated through a cAMP/PKA pathway, by either betagamma-subunit activation of an adenylyl cyclase type II or promiscuous coupling to G(s alpha).

Xenopus laevis Oocytes Contain Endogeneous Large Conductance Ca2+-activated K+ Channels

Xenopus laevis oocytes have become a pre-eminent tool for studying cloned ion channels, primarily because they intrinsically express low levels of most types of ion channels. However, when these cells are used for single channel studies, it is essential to determine whether or not oocytes contain even low levels of endogenous ion channels with properties similar to the channel being investigated. We show here that X. laevis oocytes express endogenous large-conductance Ca2+-activated K+ channels with properties similar to mammalian isoforms of this channel. The endogenous channels exhibit a voltage-dependence of 12–14 mV per e-fold change in open probability (po), can be activated by micromolar Ca2+ concentrations, and have a single channel conductance of approximately 200 pS in symmetrical 110 mM K+ solutions. Patch clamp experiments indicate that this endogenous channel is present at low densities (∼1 channel/3000 μm2). If endogenous channel subunits can form functional tetramers with other exogenous potassium channel subunits, then they will give rise to the expression of a heterogeneous channel population. Therefore, studies involving the heterologous expression of large-conductance Ca2+-activated K+ channels in Xenopus laevis oocytes require careful analysis and interpretation. Copyright © 1996 Elsevier Science Ltd

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.