Tethered Spectroscopic Probes Estimate Dynamic Distances with Subnanometer Resolution in Voltage-Dependent Potassium Channels

Measurements of inter- and intramolecular distances are important for monitoring structural changes and understanding protein interaction networks. Fluorescence resonance energy transfer and functionalized chemical spacers are the two predominantly used strategies to map short-range distances in living cells. Here, we describe the development of a hybrid approach that combines the key advantages of spectroscopic and chemical methods to estimate dynamic distance information from labeled proteins. Bifunctional spectroscopic probes were designed to make use of adaptable-anchor and length-varied spacers to estimate molecular distances by exploiting short-range collisional electron transfer. The spacers were calibrated using labeled polyproline peptides of defined lengths and validated by molecular simulations. This approach was extended to estimate distance restraints that enable us to evaluate the resting-state model of the Shaker potassium channel.     

Three Types of Single Voltage-Dependent Potassium Channels in the Sarcolemma of Frog Skeletal Muscle

Patch-clamp experiments in the sarcolemma of frog skeletal muscle evidenced the presence of three types of voltage-dependent single-channel K⁺ currents. According to their unitary conductance at a membrane voltage of +40 mV, we classified them as 16-, 13-, and 7-pS K⁺ channels. The 16-pS K⁺ channels are active close to a membrane voltage of −80 mV and they do not become inactivated during voltage pulses of 100 ms. Within 10 min after beginning the recording, these channels developed rundown with an exponential time course. The 13-pS K⁺ channels are active near −60 mV; upon a 100-ms depolarization, they exhibited inactivation with an approximate exponential time course. The 7-pS K⁺ channels were recorded at voltages positive to 0 mV. In patches containing all three types of K⁺ channels, the ensemble average currents resemble the kinetic properties of the macroscopic delayed rectifier K⁺ currents recorded in skeletal muscle and other tissues. In conclusion, the biophysical properties of unitary K⁺ currents suggest that these single-channel K⁺ currents may underlie the macroscopic delayed K⁺ currents in frog skeletal muscle fibers. In addition, since the 16- and 13-pS channels were more frequently recorded, both are the main contributors to the delayed K⁺ currents.     

Differential Regulation of Calcium-Activated Potassium Channels by Dynamic Intracellular Calcium Signals

Calcium (Ca²⁺)-activated K⁺ (K_Ca) channels regulate membrane excitability and are activated by an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), leading to membrane hyperpolarization. Most patch clamp experiments that measure K_Ca currents use steady-state [Ca²⁺] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca²⁺]_i changes dynamically, for example during [Ca²⁺]_i oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca²⁺]_i on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K_Ca subtype in isolation were used to simultaneously measure agonist-evoked [Ca²⁺]_i signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca²⁺]_i oscillations induced a corresponding K_Ca current that faithfully followed the [Ca²⁺]_i in 13–50% of cells, suggesting a good synchronization. However, [Ca²⁺]_i and K_Ca current was much less synchronized in 50–76% of cells that exhibited Ca²⁺-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca²⁺]_i events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca²⁺]_i and K_Ca current was least synchronized, 36% of total [Ca²⁺]_i spikes occurred without activating a corresponding K_Ca current spike, suggesting that BK_Ca channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca²⁺]_i and K_Ca current suggests that this relationship is more complex than could be predicted from steady-state [Ca²⁺]_i and K_Ca current. These phenomena may be important for encoding stimulus–response coupling in various cell types.     

Protein Kinase C-Mediated Down-Regulation of Voltage-Dependent Sodium Channels in Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 12- O -tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [³H]saxitoxin ([³H]STX) binding in a concentration (IC₅₀ = 19 n M )- and time ( t _1/2 = 4.5 h)-dependent manner. TPA (100 n M for 15 h) lowered the B _max of [³H]STX binding by 53% without altering the K _D value. Phorbol 12,13-dibutyrate (PDBu) also reduced [³H]STX binding, whereas 4α-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced ²²Na⁺ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced ²²Na⁺ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.     

Long-pore Electrostatics in Inward-rectifier Potassium Channels

Inward-rectifier potassium (Kir) channels differ from the canonical K⁺ channel structure in that they possess a long extended pore (~85 Å) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a K⁺ ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 Å, suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kir channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores.     

Role of Voltage-gated Potassium Channels in Cancer

Ion channels are being associated with a growing number of diseases including cancer. This overview summarizes data on voltage-gated potassium channels (VGKCs) that exhibit oncogenic properties: ether-à-go-go type 1 (Eag1). Normally, Eag1 is expressed almost exclusively in tissue of neural origin, but its ectopic expression leads to uncontrolled proliferation, while inhibition of Eag1 expression produces a concomitant reduction in proliferation. Specific monoclonal antibodies against Eag1 recognize an epitope in over 80% of human tumors of diverse origins, endowing it with diagnostic and therapeutic potential. Eag1 also possesses unique electrophysiological properties that simplify its identification. This is particularly important, as specific blockers of Eag1 currents are not available. Molecular imaging of Eag1 in live tumor models has been accomplished with dye-tagged antibodies using 3-D imaging techniques in the near-infrared spectral range. Abbreviations: EAG: Ether-à-go-go, VGKCs: voltage-gated potassium channels     

Sodium-dependent Potassium Channels in Leech P Neurons

In leech P neurons the inhibition of the Na⁺-K⁺ pump by ouabain or omission of bath K⁺ leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K⁺ and Na⁺ are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K⁺ conductance of the plasma membrane, which compensates for the reduction of the K⁺ gradient. The data presented here strongly suggest that the increased K⁺ conductance is due to Na⁺-activated K⁺ (K_Na) channels. Specifically, an increase in the cytosolic Na⁺ concentration ([Na⁺]_i) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R_in) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R_in and [Na⁺]_i followed a simple model in which the R_in decrease was attributed to K⁺ channels that are activated by the binding of three Na⁺ ions, with half-maximal activation at [Na⁺]_i between 45 and 70 mM. At maximum channel activation, R_in was reduced by more than 90%, suggesting a significant contribution of the K_Na channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K_Na channels in leech P neurons are also activated by Li⁺.     

Up-Regulation of Functional Voltage-Dependent Sodium Channels by Insulin in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [³H]saxitoxin ([³H]STX) binding in a time-dependent manner (t_1/2 = 26 h). Insulin (100 nM for 4 days) increased the B_max of [³H]STX binding by 49% without changing the K_D value and also augmented the maximal influx of ²²Na⁺ due to 560 µM veratridine by 39% without altering the EC₅₀ value of veratridine. The stimulatory effect of insulin on ²²Na⁺ influx was concentration-dependent with an EC₅₀ of 3 nM, whereas insulin-like growth factor (IGF)-I had little effect at 1 nM. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine (100 µM)-induced ²²Na⁺ influx by approximately twofold in both insulin-treated cells and untreated cells. Veratridine-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine-induced ²²Na⁺ influx via the nicotinic receptor-ion channel complex and high-K⁺ (direct activation of voltage-dependent Ca²⁺ channels)-induced ⁴⁵Ca²⁺ influx. Stimulatory effects of insulin on [³H]STX binding and veratridine-induced ²²Na⁺ influx were nullified by simultaneous treatment with either 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na⁺ channel α-subunit. These results suggest that the binding of insulin to insulin (but not IGF-I) receptors mediates the up-regulation of functional Na⁺ channel expression at plasma membranes; this up-regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).     

Role of Potassium Channels in Amyloid-Induced Cell Death

Abstract: Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid β-peptide (Aβ) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to Aβ. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K⁺ current with delayed rectifier characteristics. Increases of 64% (±19; p < 0.02) and 44% (±12; p < 0.02) in K⁺ current density were noted 6–12 and 12–18 h following the addition of Aβ to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 ± 4 and 39 ± 4% of SN56 cells were dead after 48- and 96-h exposures to Aβ, respectively. Perfusion of SN56 cells with 10–20 mM TEA blocked 71 ± 6 to 92 ± 2% of the outward currents, widened action potentials, elevated [Ca²⁺]_i, and inhibited 89 ± 14 and 68 ± 14% of the Aβ toxicity. High [K⁺]_o, which depolarizes cell membranes and increases [Ca²⁺]_i, also protected SN56 cells from Aβ toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K⁺ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, Aβ was toxic to a dopaminergic cell line (MES23.5) that expressed a K⁺ current with delayed rectifier characteristics; K⁺ current density was not altered by Aβ and MES23.5 cells were not protected by TEA from Aβ toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K⁺ currents was resistant to the toxicity of Aβ. These data suggest that a K⁺ channel with delayed rectifier characteristics may play an important role in Aβ-mediated toxicity for septal cholinergic cells.     

Properties of Shaker-type Potassium Channels in Higher Plants

Potassium (K⁺), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na⁺/K⁺ exchanger, which widely exists in animal cells, K⁺ channels and some type of K⁺ transporters function as K⁺ uptake systems in plants. Plant voltage-dependent K⁺ channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K⁺ channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K⁺ channels have been identified and play a crucial role in K⁺ homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K⁺ channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K⁺ channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K⁺ channels in plants in comparison to those of Shaker channels in animals and bacteria.     

Serotonergic Agonists Inhibit Calcium-Activated Potassium and Voltage-Dependent Sodium Currents in Rat Taste Receptor Cells

Recently we reported that rat taste receptor cells respond to the neurotransmitter serotonin with an inhibition of a calcium-activated potassium current [17]. In the present study, this observation is confirmed and extended by studying the effects of an array of serotonergic agonists on membrane properties, calcium-activated potassium current, and voltage-dependent sodium current in taste receptor cells using the patch-clamp recording technique in the whole-cell configuration. Serotonergic inhibition of calcium-activated potassium current was mimicked by the agonists N-(3-trifluoromethylphenyl)piperazine and by (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Both produced reversible inhibition of K _Ca as well as significantly increasing the input resistance of the cell. The agonists 1-(1-naphthyl)piperazine and buspirone (both serotonin receptor 1A agonists) were similarly effective in reducing K _Ca . Outward current was unaffected by application of phenylbiguanide, a serotonin receptor 3 agonist, though current was affected by subsequent application of (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Two agonists—N-(3-trifluoromethylphenyl)piperazine and (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene—were also tested on voltage-dependent sodium currents; both were effective and reversible in reducing its magnitude at a variety of applied potentials. These data are consistent with the notion that serotonin effects in rat taste receptor cells are mediated by serotonin 1A receptors, though other receptor subtypes may be additionally expressed. Serotonin may affect the taste cell electrical properties during active stimulation in a paracrine fashion. Received: 10 May 1999/Revised: 27 September 1999     

心肌细胞钾通道的研究进展

从分子水平上看,所有钾通道都是由一个基因家族中的基因所编码的４个亚基组成,通道的失活门控机制在Ｎ型、Ｃ型和Ｐ型三种,通道的孔道结构均在跨膜片段Ｓ５至Ｓ６之间,尽管各种钾通道在分子结构上的共处远多于不同之处,但每种钾通道的个性表现却有非常重要的生理意义,迄今为止在心肌细胞上发现的８种钾通道的电导值,门控动力学特征、离子动力学特征,通道激动剂,阻断剂和调制剂均不同。     

Transient Cytoplasmic pH Changes in Correlation with Opening of Potassium Channels in Eremosphaera

Steigner, W. Khler, K., Simonis, W. and Urbach, W. 1988. Transientcytoplasmic pH changes in correlation with opening of potassiumchannels in Eremosphaera.—J. exp. Bot. 39: 23–36. The role of the cytoplasmic pH (pH_c) of Eremosphaera viridisin the signal transduction chain after light-off from the chloroplaststo the K⁺ channels in the plasmalemma of this unicellular algawas investigated. The temporary opening of K⁺ channels is indicatedby a transient hypcrpolarization (TP). To record rapid changesof pH_c, continuous measurements with pH sensitive micro-electrodeswere carried out. (i) Under normal conditions pH_c in the light(7·56 ±0·2) did not differ from pHc inthe dark (7·62 ±0·2). (ii) The vacuolepH ranged between 4·8 and 5·2. (iii) After light-offa rapid transient acidification of pHc O19±0·07occurred and a TP was released, (iv) In every case, the startof the transient acidification after light-off preceded thehyperpolarization by about 3s. (v) Light-on caused a rapid transientalkalinization but never a TP. (vi) Change to acid externalmedium (3.2) transiently acidified the cytoplasm and was ableto release a TP. (vii) After addition of NH₄Cl, pH_c again showeda rapid transient acidification and the release of a TP. The origin of the protons appearing in the cytoplasm after light-offis discussed critically with respect to the buffer capacity.Either direct or indirect translocation is a possible mechanismfor the movement of H⁺ from the chloroplasts into the cytoplasm.The intracellular acidification and its relation to the openingof potassium channels in the plasmalemma leads us to suggestthat a sudden change of pH_c is a potent internal signal factorin Eremosphaera viridis. Key words: Cytoplasmic pH, transient potential, K⁺–channels, Eremosphaera viridis     

Hormonal and Neurotransmitter Regulation of Ca++ Influx Through Voltage-Dependent Slow Channels in Cardiac Muscle Membrane

The voltage-and time-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca⁺⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. These slow channels behave kinetically as if their gates open, close, and recover more slowly than those of the fast Na⁺ channels; in addition, the slow channel gates operate over a less negative (more depolarized) voltage range. Tatrodotoxin does not block the slow channels, whereas the calcium antagonistic drugs, Mn⁺⁺, Co⁺⁺, and La⁺⁺⁺ ions do. The slow channels have some special properties, including their functional dependence on metabolic energy, their selective blockade by acidosis, and their regulation by cyclic AMP level. Because of their regulation by cyclic AMP, it is proposed that either the slow channel protein or an associated regulatory protein must be phosphorylated in order for the channel to be made available for voltage activation during excitation. That is, the dephosphorylated channel would be electrically silent.

The requirement for phosphorylation allows the extrinsic control of the slow channels and Ca⁺⁺ influx by neurotransmitters, hormones, and autacoids that affect the cyclic nucleotide levels.     

Pore Hydration States of KcsA Potassium Channels in Membranes

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes, but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the transmembrane domain of M2 protein of influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid state NMR. In contrast, the M2 proton channel shows a V-shaped water profile across the membrane, with a narrow constriction at the center, like the hourglass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.     

Interaction of Tetraethylammonium Ion Derivatives with the Potassium Channels of Giant Axons

A number of compounds related to TEA⁺ (tetraethylammoniumion) were injected into squid axons and their effects on g_K (the potassium conductance) were determined. In most of these ions a quaternary nitrogen is surrounded by three ethyl groups and a fourth group that is very hydrophobic. Several of the ions cause inactivation of g_K, a type of ionic gating that is not normally seen in squid axon; i.e., after depolarization g_K increases and then spontaneously decreases to a small fraction of its peak value even though the depolarization is maintained. Observations on the mechanism of this gating show that (a) QA (quaternary ammonium) ions only enter K⁺ channels that have open activation gates (the normal permeability gates). (b) The activation gates of QA-occluded channels do not close readily. (c) Hyperpolarization helps to clear QA ions from the channels. (d) Raising the external K⁺ concentration also helps to clear QA ions from the channels. Observations (c) and (d) strongly suggest that K⁺ ions traverse the membrane by way of pores, and they cannot be explained by the usual type of carrier model. The data suggest that a K⁺ pore has two distinct parts: a wide inner mouth that can accept a hydrated K⁺ ion or a TEA⁺-like ion, and a narrower portion that can accept a dehydrated or partially dehydrated K⁺ ion, but not TEA⁺.     

Regulated RNA Editing and Functional Epistasis in Shaker Potassium Channels

Regulated point modification by an RNA editing enzyme occurs at four conserved sites in the Drosophila Shaker potassium channel. Single mRNA molecules can potentially represent any of 2⁴ = 16 permutations (isoforms) of these natural variants. We generated isoform expression profiles to assess sexually dimorphic, spatial, and temporal differences. Striking tissue-specific expression was seen for particular isoforms. Moreover, isoform distributions showed evidence for coupling (linkage) of editing sites. Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells. Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites. Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.     

Stretch-Activated Potassium Channels in Hypotonically Induced Blebs of Atrial Myocytes

Stress in the lipids of the cell membrane may be responsible for activating stretch-activated channels (SACs) in nonspecialized sensory cells such as cardiac myocytes, where they are likely to play a role in cardiac mechanoelectric feedback. We examined the influence of the mechanical microenvironment on the gating of stretch-activated potassium channels (SAKCs) in rat atrial myocytes. The goal was to examine the role of the cytoskeleton in the gating process. We recorded from blebs that have minimal cytoskeleton and cells treated with cytochalasin B (cyto-B) to disrupt filamentous actin. Histochemical and electron microscopic techniques confirmed that the bleb membrane was largely free of F-actin. Channel currents showed mechanosensitivity and potassium selectivity and were activated by low pH and arachidonic acid, similar to properties of TREK-1. Some patches showed a time-dependent decrease in current that may be adaptation or inactivation, and since this decrease appeared in control cells and blebs, it is probably not the result of adaptation in the cytoskeleton. Cyto-B treatment and blebbing caused an increase in background channel activity, suggesting a transfer of stress from actin to bilayer and then to the channel. The slope sensitivity of gating before and after cyto-B treatment was similar to that of blebs, implying the characteristic change of dimensions associated with channel gating was the same in the three mechanical environments. The mechanosensitivity of SAKCs appears to be the result of interaction with membrane lipids and not of direct involvement of the cytoskeleton.