Differential blockage of two types of potassium channels in the crab giant axon

Summary Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crabsCarcinus maenas andCancer pagirus. The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate types of potassium channels exist in these axons (M. E. Quinta-Ferreira, E. Rojas and N. Arispe,J. Membrane Biol. 66:171–181, 1982). It is shown here that, with small concentrations of conventional K⁺-channel blockers, it is possible to differentially inhibit these channels. The potassium channels with activation and fast inactivation gating (m³h, Hodgkin-Huxley kinetics) were blocked by external application of 4 amino-pyridine (4-AP). The potassium channels with standard gating (n⁴, Hodgkin-Huxley kinetics) were preferentially inhibited by externally applied tetraethylammonium (TEA). The differential blockage of the two types of potassium conductance changes suggests that they represent two different populations of potassium channels.It is further shown here that blocking the early transient conductance increase leads to the inhibition of the repetitive electrical activity induced by constant depolarizing current injection in fibers fromCardisoma guanhumi.     

Potassium currents in the giant axon of the crab Carcinus maenas

Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crab Carcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol, (London) 143:76-90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using the m3h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standard n4 Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

Ion conductance and ion selectivity of potassium channels in snail neurones

Summary Delayed potassium channels were studied in internally perfused neurone somata from land snails. Relaxation and fluctuation analysis of this class of ion channels revealed Hodgkin-Huxley type K channels with an average single channel conductance ( _K) of 2.40±0.15 pS. The conductance of open channels is independent of voltage and virtually all K channels seem to be open at maximum K conductance (g _K) of the membrane. Voltage dependent time constants of activation ofg _K, calculated from K current relaxation and from cut-off frequencies of power spectra, are very similar indicating dominant first-order kinetics. Ion selectivity of K channels was studied by ion substitution in the external medium and exhibited the following sequence: T1⁺>K⁺>Rb⁺>Cs⁺>NH ₄ ⁺ >Li⁺>Na⁺. The sequence of the alkali cations does not conform to any of the sequences predicted by Eisenman's theory. However, the data are well accommodated by a new theory assuming a single rate-limiting barrier that governs ion movement through the channel.This paper is dedicated to the memory of Walther Wilbrandt.     

Role of hydrophobic and ionic forces in the movement of S4 of the Shaker potassium channel

Abstract

Voltage-gated ion (K⁺, Na⁺, Ca²⁺) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd²⁺, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.     

Electrophysiological properties of cellular and paracellular conductive pathways of the rabbit cortical collecting duct

Summary Microelectrode techniques were applied to the rabbit isolated perfused cortical collecting duct to provide an initial quantitation and characterization of the cell membrane and tight junction conductances. Initial studies demonstrated that the fractional resistance (ratio of the resistance of the apical cell membrane to the sum of the resistances of the apical and basolateral membranes) was usually independent of the point along the tubule of microelectrode impalement—implicating little cell-to-cell coupling—supporting the application of quantitative techniques to the cortical collecting duct. It was demonstrated that in the presence of amiloride, either reduction in the luminal pH or the addition of barium to the perfusate selectively reduced the apical membrane potassium conductance. From the changes inG ^te and fractional resistance upon reducing the luminal pH or addition of barium to the perfusate, the transepithelial, apical membrane, basolateral membrane and tight junction conductances were estimated to be 9.3, 6.7, 8.1 and 6.0 mS cm^–2, respectively. Ninety to ninety-five percent of the apical membrane conductance reflected the barium-sensitive potassium conductance in the presence of amiloride with an estimated potassium permeability of 1.1×10^–4 cm sec^–1. Reduction in the perfusate pH to 4.0 caused a 70% decrease in the apical membrane potassium conductance, implying a blocking site with an acidic group having a pK _a near 4.4. It is concluded that both the transcellular and paracellular pathways of the cortical collecting tubule have high ionic conductances, and that the apical membrane conductance primarily reffects a high potassium conductance. Furthermore, both reduction in the perfusate pH and addition of barium to the perfusate selectively block the apical potassium channels, although the site of inhibition likely differs since the two ions display markedly different voltage-dependent blocks of the channel.     

Conditioning prepulses and kinetics of potassium conductance in the frog node

Summary The kinetics of potassium conductance were analyzed in response to voltage-clamp steps with holding potential (–75 mV) as initial condition and after a positive prepulse to-wards +45 mV of 10-msec duration. As the potassium reversal potentialE _K altered during potassium current flow, a method to obtain the conductance independent ofE _K was used. Conductance kinetics at 15°C were analyzed according to the Hodgkin-Huxley (HH) model. The time constant of potassium activation, with holding potential as initial condition, is a monotonous decreasing function of membrane potential. Its value ofca. 9 msec at –50 mV decreases to 1 msec at +30 mV. Changes inE _K did not affect the voltage dependency of this time constant. The time constant of potassium deactivation, i.e. the off-response following a 10-msec prepulse towards +45 mV, shows a completely different voltage dependency. At a membrane potential of –90 mV it is approximately 2 msec and gradually increases for more positive voltages towards a maximum value of about 6 msec, that is reached between –5 and 0 mV. At still larger values of membrane voltage this time constant starts to fall again. It is concluded that a HH-model, as applied for a single population of potassium channels, has to be rejected. Computer simulations indicate that an extension to two populations of independent potassium channels, each with HH-kinetics, is also inconsistent with the observed results.     

Apical Nonspecific Cation Channels in Everted Collecting Tubules of Potassium-Adapted Ambystoma

We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water. Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels. In cell-attached patches the conductance averaged 30.3 ± 2.4 (9) pS. Since replacement of the chloride in the patch pipette with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the current reversed (movement of potassium into the pipette) was −29.6 ± 6.5(9) mV. Open probability at 0 mV pipette voltage was 0.08 ± 0.03 and was unaffected when the apical membrane was exposed to either 2 × 10⁻⁶ or 2 × 10⁻⁵ m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 ± 0.002 without affecting the conductance of the apical channel. These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium. Received: 14 January 2000/Revised: 16 June 2000     

Potassium conductance models related to an interactive subunit membrane

Summary The properties of various potassium conductance models have been investigated using an analogue computer. It is shown that the experimental data of Hodgkin and Huxley can be fitted as satisfactorily by a cube (n ³) model of potassium conductance as it is by the Hodgkin-Huxley (n ⁴) and (n ⁶) models.A planar subunit array structure for the membrane has been suggested, where the appearance of a potassium conducting channel depends upon a conformational change to an activated state in each of neighboring subunits. This system is described by the same mathematics as the Hodgkin-Huxley activating particle mechanism and so provides a physical basis for the power (n ) formulae. Introduction of interaction between subunits, such that a conformational change is prohibited unless an adjacent subunit is in the activated state, modifies the mathematics and enables simulation of the delayed potassium currents observed by Cole and Moore (Biophys. J. 1:1, 1960). This innovation avoids the difficulties associated with the higher power (>6) models, by not requiring physical justification for large numbers of simultaneous events, while still providing a good fit to the experimental data. The interactive subunit models satisfactorily describe the potassium conductance changes which occur under voltage clamp or during an action potential.A preliminary account of this work was presented at the Seventh Annual Meeting ofPhysics in Medicine and Biology, Biophysics Group, Australian Institote of Physics, Adelaide, South Australia, May 1967.     

Characterization of a stretch‐activated potassium channel in chondrocytes

Chondrocytes possess the capacity to transduce load‐induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch‐clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell‐attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm²). The mean slope conductance of the principal single channels resolved within the total stretch‐activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, E_K+, suggesting it was a high‐conductance potassium channel. Activation of these high‐conductance potassium channels was inhibited by extracellular TEA (K_d approx. 900 µM) and iberiotoxin (K_d approx. 40 nM). This suggests that the current was largely carried by BK‐like potassium (MaxiK) channels. To further characterize these BK‐like channels, we used inside‐out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1‐ and β1‐subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction. J. Cell. Physiol. 223: 511–518, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence that potassium channels mediate the effects of serotonin on the ocular circadian pacemaker of Aplysia

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Serotonin is known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate whether potassium channels are involved in effects on the ocular circadian rhythm. Our experimental approach was to study the effect of the potassium channel antagonist barium on serotonin-induced phase shifts of this rhythm. The application of barium was found to block serotonininduced phase shifts whereas barium alone did not cause significant phase shifts. The effects of barium were found to be dose dependent. In addition, barium blocked forskolin-induced phase advances but did not interfere with serotonin-induced increases in cAMP content. Finally, barium antagonized serotonin-induced suppression of compound action potential activity. These results are consistent with a model in which the application of serotonin phase shifts the ocular pacemaker by causing a membrane hyperpolarization which is mediated by a cAMP-dependent potassium conductance.Abbreviations ASW artificial seawater - Ba^{+ +} barium - CAP compound action potential - CT circadian time - 5-HT serotonin - TEA tetraethylammonium     

ATP-dependent potassium channels of muscle cells: Their properties,regulation, and possible functions

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a lowP _open at physiological [ATP]_i. The unitary conductance is 15–20 pS at physiological [K⁺] _o , and the channels are highly selective for K⁺. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. K_ATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pH_i. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and closed states. The main effect of ATP is to increase occupancy of long-lived closed states. The channels may have a role in the control of excitability and probably act as a route for K⁺ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K⁺ selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of ⁸⁶Rb⁺ into chromaffin granules prepared from bovine adrenal gland medulla. The ⁸⁶Rb⁺ influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432±9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca²⁺-activated K⁺ channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL ⁻ knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL ⁺ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.     

How Does the W434F Mutation Block Current in Shaker Potassium Channels?

The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH₂-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10⁻⁵ or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.     

Reduced transmitter release conferred by mutations in theslowpoke-encoded Ca2+-activated K+ channel gene ofDrosophila

Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission. Here we describe the effects of mutations in theslowpoke gene, which is the structural gene for a calcium activated potassium channel, on transmitter release at the neuromuscular junction inDrosophila melanogaster. Surprisingly, we find that theslowpoke mutant exhibits reduced transmitter release compared to normal. Similarly, theslowpoke mutation significantly suppresses the increased transmitter release conferred either by a mutation inShaker or by application of 4-aminopyridine, which blocks theShaker-encoded potassium channel at theDrosophila nerve terminal. Furthermore, theslowpoke mutation suppresses the striking increase in transmitter release that occurs following application of 4-aminopyridine to theether a go-go mutant. This suppression is most likely the result of a reduction of Ca²⁺ influx into the nerve terminal in theslowpoke mutant. We hypothesize that the effects of theslowpoke mutation are indirect, perhaps resulting from increased Ca²⁺ channel inactivation, decreased Na⁺ or Ca²⁺ channel localization or gene expression, or by increases in the expression or activity of potassium channels distinct fromslowpoke.     

Properties of single potassium channels in guinea pig hepatocytes

The patch-clamp technique of cell-attached and inside-out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K⁺ concentrations. A linear single-channel current-voltage relationship was obtained at the voltage range of -80 to -20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K⁺ concentration of 148 mM in the cell-attached patch membrane, the I-V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K⁺]₀) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K⁺]₀, indicating that this channel was highly selective for K⁺. Open probabilities (P₀) of the channel were 73-93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K⁺ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34-36°C. P₀ in inside-out patches was not changed by application of 1 μM Ca²⁺ nor 1 mM Mg²⁺ to the internal side of patch membranes. It is concluded that a novel type of the K⁺ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca²⁺]_i, from those in other species. © 1994 Wiley-Liss, Inc.     

Modulation of the mitochondrial large-conductance calcium-regulated potassium channel by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) and their metabolites can modulate several biochemical processes in the cell and thus prevent various diseases. PUFAs have a number of cellular targets, including membrane proteins. They can interact with plasma membrane and intracellular potassium channels. The goal of this work was to verify the interaction between PUFAs and the most common and intensively studied mitochondrial large conductance Ca²⁺-regulated potassium channel (mitoBK_Ca). For this purpose human astrocytoma U87 MG cell lines were investigated using a patch-clamp technique. We analyzed the effects of arachidonic acid (AA); eicosatetraynoic acid (ETYA), which is a non-metabolizable analog of AA; docosahexaenoic acid (DHA); and eicosapentaenoic acid (EPA). The open probability (P_o) of the channel did not change significantly after application of 10 μM ETYA. P_o increased, however, after adding 10 μM AA. The application of 30 μM DHA or 10 μM EPA also increased the P_o of the channel. Additionally, the number of open channels in the patch increased in the presence of 30 μM EPA. Collectively, our results indicate that PUFAs regulate the BK_Ca channel from the inner mitochondrial membrane.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.