Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics

Genistein, a generic tyrosine kinase inhibitor, has been used extensively as a tool to investigate the possible regulation of membrane function by tyrosine phosphorylation. Genistein, in micromolar concentrations, alters the function of numerous ion channels and other membrane proteins, but only in few cases has it been demonstrated that the changes in membrane protein (ion channel) function are due to changes in a protein's phosphorylation status. The major common denominator characterizing proteins that are modulated by genistein seems to be that they are imbedded into, and span, the bilayer component of the plasma membrane. We therefore explored whether genistein could alter ion channel function by a bilayer-mediated mechanism and examined genistein's effect on gramicidin A (gA) channels in planar phospholipid bilayers. gA channels form by transmembrane dimerization of two nonconducting subunits, and genistein potentiates gA channel activity by increasing the appearance rate and prolonging the lifetime of bilayer-spanning gA dimers. That is, genistein shifts the equilibrium between nonconducting monomers and conducting dimers in favor of the bilayer-spanning dimers; the changes in channel activity therefore cannot be due to changes in bilayer fluidity. To obtain further insights into the mechanism underlying this modulation of gA channel function, we examined the effects of genistein on channels formed by gA analogues that differ in amino acid sequence. For a given channel length, the effects of genistein on gA dimerization do not depend on the specific sequence, or the chirality, of the channel-forming gA analogues. In contrast, when we change the channel length (by decreasing or increasing the number of amino acid residues in the sequence), or the bilayer thickness (by changing methylene groups in the acyl chains), the magnitude of genistein's effect increases with increasing hydrophobic mismatch between the channel length and the bilayer thickness. These results strongly suggest that genistein alters bilayer mechanical properties, which in turn modulates channel function. This bilayer-mediated mechanism is likely to apply to other pharmacological reagents and membrane proteins.     

Concanavalin A modulates a potassium channel in cultured Aplysia neurons

A novel 100 pS K(+)-selective ion channel is frequently observed in cell-attached membrane patches from cultured Aplysia neurons. The activity of this channel is moderately voltage-dependent, but channel openings are rare and brief even when the patch is strongly depolarized. However, the activity of the channel is increased dramatically by the addition of the lectin concanavalin A (Con A), to the patch pipette. The channel is also activated by Con A in the bathing medium, suggesting that the lectin's action is via an as yet unidentified intracellular second messenger. In the one single-channel patch studied, Con A had no effect on the channel mean open time; rather it decreased the average duration of the long closed times between bursts of openings. Thus Con A increases either the open probability of single channels, the number of functional channels in the patch, or both. The functional significance of the Con A-induced modulation of K+ channel activity remains to be determined.     

Ionic channels: modulation by G proteins and by phosphorylation.

The gating of ion channels may be modulated by G proteins or by phosphorylation. Direct coupling between G proteins and ion channels has been shown in excised patches of membrane. Steps must now be taken to study the protein domains of G proteins and ion channels involved in the mutual interaction. The concept of channel modulation by protein kinases has recently been extended to include additional types of ion channel.     

Neuromodulation of Na+ channels: an unexpected form of cellular plasticity

Voltage-gated Na+ channels set the threshold for action potential generation and are therefore good candidates to mediate forms of plasticity that affect the entire neuronal output. Although early studies led to the idea that Na+ channels were not subject to modulation, we now know that Na+ channel function is affected by phosphorylation. Furthermore, Na+ channel modulation is implicated in the control of input-output relationships in several types of neuron and seems to be involved in phenomena as varied as cocaine withdrawal, hyperalgesia and light adaptation. Here we review the available evidence for the regulation of Na+ channels by phosphorylation, its molecular mechanism, and the possible ways in which it affects neuronal function.     

Rundown of the hyperpolarization-activated KAT1 channel involves slowing of the opening transitions regulated by phosphorylation.

Disappearance of the functional activity or rundown of ion channels upon patch excision in many cells involves a decrease in the number of channels available to open. A variety of cellular and biophysical mechanisms have been shown to be involved in the rundown of different ion channels. We examined the rundown process of the plant hyperpolarization-activated KAT1 K+ channel expressed in Xenopus oocytes. The decrease in the KAT1 channel activity on patch excision was accompanied by progressive slowing of the activation time course, and it was caused by a shift in the voltage dependence of the channel without any change in the single-channel amplitude. The single-channel analysis showed that patch excision alters only the transitions leading up to the burst states of the channel. Patch cramming or concurrent application of protein kinase A (PKA) and ATP restored the channel activity. In contrast, nonspecific alkaline phosphatase (ALP) accelerated the rundown time course. Low internal pH, which inhibits ALP activity, slowed the KAT1 rundown time course. The results show that the opening transitions of the KAT1 channel are enhanced not only by hyperpolarization but also by PKA-mediated phosphorylation.     

Zipper transition in an alpha-helix: a mechanism for gating of voltage-sensitive ion channels in a biological membrane

It is suggested that the gating currents which control the ion channels in a biological membrane are comprised of positive charges crossing the membrane along chains of hydrogen bonds. These chains are the sets of hydrogen bonds which hold alpha-helical protein segments in their rigid conformations. The passage of a positive charge in one direction along such a chain will convert hydrogen bonds from the usual rigid N--C = O...H--N form to a flaccid N = C--O--H...N form. This "zipper" transition can be reversed by the passage of the positive charge along the return route. A flaccid protein rod can clog an ion channel and thereby close it. When all of the protein rods framing an ion channel are in the rigid conformation, the channel is open. This mechanism is used to explain some of the observed characteristics of calcium ion channels and sodium ion channels.     

Phosphorylation of K+ channels in the squid giant axon. A mechanistic analysis

Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In squid giant axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.     

Stretch-activation and stretch-inactivation of Shaker-IR, a voltage-gated K+ channel

Mechanosensitive (MS) ion channels are ubiquitous in eukaryotic cell types but baffling because of their contentious physiologies and diverse molecular identities. In some cellular contexts mechanically responsive ion channels are undoubtedly mechanosensory transducers, but it does not follow that all MS channels are mechanotransducers. Here we demonstrate, for an archetypical voltage-gated channel (Shaker-IR; inactivation-removed), robust MS channel behavior. In oocyte patches subjected to stretch, Shaker-IR exhibits both stretch-activation (SA) and stretch-inactivation (SI). SA is seen when prestretch P(open) (set by voltage) is low, and SI is seen when it is high. The stretch effects occur in cell-attached and excised patches at both macroscopic and single-channel levels. Were one ignorant of this particular MS channel's identity, one might propose it had been designed as a sophisticated reporter of bilayer tension. Knowing Shaker-IR's provenance and biology, however, such a suggestion would be absurd. We argue that the MS responses of Shaker-IR reflect not overlooked "mechano-gating" specializations of Shaker, but a common property of multiconformation membrane proteins: inherent susceptibility to bilayer tension. The molecular diversity of MS channels indicates that susceptibility to bilayer tension is hard to design out of dynamic membrane proteins. Presumably the cost of being insusceptible to bilayer tension often outweighs the benefits, especially where the in situ milieu of channels can provide mechanoprotection.     

Combined transport of water and ions through membrane channels

The coupling of ion and water flow through membrane channels is under dispute. Among all human aquaporins only aquaporin-6 exhibits ion channel activity. Whether aquaporin-6 functions also as a water channel cannot yet be determined with confidence. Similarly, a comparison of single-channel water permeabilities from ion channels and aquaporins suggests that ion channels may play a secondary role as water channels. However, the fraction of absorbed fluid that crosses epithelial ion channels still remains to be determined.     

Counting channels: a tutorial guide on ion channel fluctuation analysis

Ion channels open and close in a stochastic fashion, following the laws of probability. However, distinct from tossing a coin or a die, the probability of finding the channel closed or open is not a fixed number but can be modified (i.e., we can cheat) by some external stimulus, such as the voltage. Single-channel records can be obtained using the appropriate electrophysiological technique (e.g., patch clamp), and from these records the open probability and the channel conductance can be calculated. Gathering these parameters from a membrane containing many channels is not straightforward, as the macroscopic current I = iNP(o), where i is the single-channel current, N the number of channels, and P(o) the probability of finding the channel open, cannot be split into its individual components. In this tutorial, using the probabilistic nature of ion channels, we discuss in detail how i, N, and P(o max) (the maximum open probability) can be obtained using fluctuation (nonstationary noise) analysis (Sigworth FJ. G Gen Physiol 307: 97-129, 1980). We also analyze the sources of possible artifacts in the determination of i and N, such as channel rundown, inadequate filtering, and limited resolution of digital data acquisition by use of a simulation computer program (available at www.cecs.cl).     

Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore.

Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes

CRAC channels are key calcium conduits in both physiological and pathological states. Understanding how these channels are controlled is important as this will not only provide insight into a novel signal transduction pathway coupling intracellular stores to the channels in the plasma membrane, but might also be of clinical relevance. Determining the molecular identity of the CRAC channels will certainly be a major step forward. Like all Ca(2+)-selective channels, CRAC channels lose their selectivity in divalent-free external solution to support large monovalent Na(+) currents. This approach has provided new insight into channel permeation and selectivity, and identifies some interesting differences between CRAC channels and voltage-operated calcium channels (VOCCs). Studies in divalent-free solution are a double-edged sword, however. Electrophysiologists need to be wary because some of the conditions used to study I(CRAC) in divalent-free external solution, notably omission of Mg(2+)/Mg-ATP from the recording pipette solution, activates an additional current permeating through Mg(2+)-nucleotide-regulated metal ion current (MagNuM; TRPM7) channels. This channel underlies the large single-channel events that have been attributed to CRAC channels in the past and which have been used to as a tool to identify store-operated channels in native cells and recombinant expression systems.Are we any closer to identifying the elusive CRAC channel gene(s)? TRPV6 seemed a very attractive candidate, but one of the main arguments supporting it was a single-channel conductance in divalent-free solution similar to that for CRAC reported under conditions where MagNuM is active. We now know that the conductance of TRPV6 is approximately 200-fold larger than that of CRAC in native tissue. Moreover, it is unclear if TRPV6 is store-operated. Further work on TRPV6, particularly whether its single-channel conductance is still high under conditions where it apparently forms multimers with endogenous store-operated channels, and whether it is activated by a variety of store depletion protocols, will be helpful in finally resolving this issue.     

Amphotericin B channels in the bacterial membrane: role of sterol and temperature

Amphotericin B is an antibiotic that forms ion channels in the membrane of a host cell. The change in permeability produced by these channels is greatly improved by sterols; nevertheless, the single channel conductivity remains invariant. Hence, it is proposed that sterols do not act directly, but rather through the modulation of the membrane phase. We look at the formation of these channels in the bacterial membrane to determine the mechanism of its known antibiotic resistance. We found that channels can indeed be formed in this membrane, but a substantial amount of amphotericin B is required. We also study the effects of the antibiotic concentration needed for channel expression as well as the dynamics of channels affected by both sterol and temperature in phosphatidylcholine membranes. The results support the idea that membrane structure is a determining factor in the action of the antibiotic.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.     

Colicin N forms voltage- and pH-dependent channels in planar lipid bilayer membranes

The protein antibiotic colicin N forms ion-permeable channels through planar lipid bilayers. Channels are induced when positive voltages higher than +60 mV are applied. Incorporated channels activate and inactivate in a voltage-dependent fashion. It is shown that colicin N undergoes a transition between an “acidic” and a “basic” channel form which are distinguishable by different voltage dependences. The single-channel conductance is non-ohmic and strongly dependent on pH, indicating that titratable groups control the passage of ions through the channel. The ion selectivity of colicin N channels is influenced by the pH and the lipid composition of the bilayer membrane. In neutral membranes the channel undergoes a transition from slightly cation-selective to slightly anion-selective when the pH is changed from 7 to 5. In lipid membranes bearing a negative surface charge the channel shows a more pronounced cation selectivity which decreases but does not reverse upon lowering the pH from 7 to 5. The high degree of similarity between the channel characteristics of colicin A and N suggests that the channels share common features in their molecular structure. Offprint requests to: F. Pattus     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Gadolinium-sensitive, voltage-dependent calcium release channels in the endoplasmic reticulum of a higher plant mechanoreceptor organ.

The lipid bilayer technique was adapted to the functional reconstitution of ion channels from the endoplasmic reticulum of a higher plant. This was obtained at high purity from touch-sensitive tendrils of Bryonia dioica. In this preparation, a calcium-selective strongly rectifying channel is prevailing whose single-channel properties have been characterized. The single-channel conductance is 29 pS in 50 mM CaCl2. The Ca2+: K+ selectivity was determined to be approximately 6.6. The channel is voltage-gated and, more importantly, the gating voltage is strongly shifted towards more negative voltages when a transmembrane Ca2+ gradient is applied. Thus, at physiological voltages across the endoplasmic reticulum membrane, the channel's open probability will be governed largely by the chemical potential gradient of Ca2+, generated by the Ca(2+)-ATPase in that same membrane. The calcium release channel described here is effectively blocked by Gd3+ which also completely suppresses a tendril's reaction to touch, suggesting that this channel could be a key element of calcium signaling in higher plant mechanotransduction. Its molecular characteristics and inhibitor data show it to be the first known member of a hitherto unrecognized class of calcium channels.