Phe-Met-Arg-Phe-amide Activates a Novel Voltage-dependent K+ Current through a Lipoxygenase Pathway in Molluscan Neurones

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED₅₀ = 23 nM) activated a K⁺ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K⁺]_o = 1.7 mM), only outward current responses occurred. In high K⁺ salines ([K⁺]_o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K⁺. In both salines, no responses were measured below −120 mV. Between −120 mV and the K⁺ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K⁺ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K⁺]_o. Since this result was also obtained in nearly symmetrical K⁺ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K⁺ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba²⁺, and, partially, Cd²⁺ and Cs⁺. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A₂ and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K⁺ current distinguish it from other receptor-driven K⁺ currents such as the S-current- and G-protein-dependent inward rectifiers.     

HERG-like K+ Channels in Microglia

A voltage-gated K⁺ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K⁺ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K⁺ conductance–voltage relation was half maximal at −59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (−14 vs. −39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795–805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs⁺ or Ba²⁺ occurred only at millimolar concentrations, La³⁺ blocked with K _i = ∼40 μM, and the HERG-selective blocker, E-4031, blocked with K _i = 37 nM. Implications of the presence of HERG-like K⁺ channels for the ontogeny of microglia are discussed.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Selectivity Changes during Activation of Mutant Shaker Potassium Channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH₂-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e₀. The fully open T442S chimeric channel has the conductance sequence Rb⁺ > NH₄ ⁺ > K⁺. The opposite conductance sequence, K⁺ > NH₄ ⁺ > Rb⁺, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb⁺.     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

Ionic interactions of Ba2+ blockades in the MthK K+ channel

The movement and interaction of multiple ions passing through in single file underlie various fundamental K⁺ channel properties, from the effective conduction of K⁺ ions to channel blockade by Ba²⁺ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba²⁺ with permeant ions within the ion conduction pathway of the MthK K⁺ channel. We found that, as typical of K⁺ channels, the MthK channel was blocked by Ba²⁺ at the internal side, and the Ba²⁺-blocking effect was enhanced by external K⁺. We also obtained crystal structures of the MthK K⁺ channel pore in both Ba²⁺–Na⁺ and Ba²⁺–K⁺ environments. In the Ba²⁺–Na⁺ environment, we found that a single Ba²⁺ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba²⁺–K⁺ environment, Ba²⁺ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba²⁺ blockade of K⁺ channels.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K⁺ concentrations ([K⁺] _o ) between 75 and 300mm and NaCl concentrations of 60, 120, or 240mm. For several combinations of KCl and NaCl, steady-state internal potentials (V _i) were the same for different [K⁺] _o . For the range ofV _i examined, K⁺ efflux occurs principally through the K⁺ inward rectifier channels. When external K⁺ is removedV _i remains constant for 2 to 3 hr because of the high membrane conductance to Cl^–, but K⁺ efflux drops by about one order of magnitude.External Ba²⁺ in the presence or absence of external K⁺ produces an inhibition of K⁺ efflux described by a relation of the formu=(u₁/(1+C)[Ba²⁺] _o ))+u ₂, whereu is the uninhibited fraction of K⁺ efflux;u ₁, u₂ andC are constants; andu ₁+u₂=1.C depends both on [K⁺] _o andV _i. When [K⁺] _o 75mm, increasing [K⁺] _o at constantV _i reduces Ba²⁺ sensitivity. For constantV _i–30 mV, Ba²⁺ sensitivity is less when [K⁺] _o =0 than when [K⁺] _o 75mm. When [K⁺] _o =0, Ba²⁺ sensitivity decreases asV _i is made more positive. The dependence of the Ba²⁺ sensitivity onV _i at constant [K⁺] _o is greater when [K⁺] _o =0 than when [K⁺] _o 75mm.Both the activation of K⁺ efflux by external K⁺ and the Ba²⁺ inhibition of K⁺ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K⁺, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba²⁺ occupies either site, K⁺ efflux is further reduced. The reduction of Ba²⁺-sensitivity by increasing [K⁺] _o at high [K⁺] _o is attributable to the displacement of Ba²⁺ from the control sites by K⁺. The increased Ba²⁺ sensitivity produced by going from [K⁺] _o =0 to [K⁺] _o >-75mm whenV _i–30 mV is attributable to states in which Ba²⁺ occupies one site and K⁺ the other when [K⁺] _o 0. The smallerV _i dependence of the Ba²⁺ sensitivity when [K⁺] _o 75mm compared to [K⁺] _o =0 is attributable to the necessity that Ba²⁺ displace K⁺ at the control sites when [K⁺] _o is high but not when [K⁺] _o =0.     

Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria

Summary Rapid uptake of Ba²⁺ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba²⁺, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba²⁺ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba²⁺ on mechanisms involved in synthesis of ATP. Ba²⁺, which has an ionic radius similar to that of K⁺, inhibits unidirectional K⁺ flux into respiring rat liver mitochondria. This effect on K⁺ influx is observable at concentrations of Ba²⁺, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba²⁺ decreases the measuredV _max of K⁺ influx, while having little effect on the apparentK _m for K⁺. The inhibition of K⁺ influx by Ba²⁺ is seen in the presence and absence of mersalyl, an activator of K⁺ influx. In contrast, under the conditions studied, Ba²⁺ has no apparent effet on the rate of unidirectional K⁺ efflux. These data are consistent with the idea that K⁺ may enter and leave mitochondria via spearate mechanisms.     

Blockade of cardiac outwardly rectifying K+ channels by TEA and class III antiarrhythmics — evidence against a single drug-sensitive channel site

Elementary K⁺ currents through cardiac outwardly rectifying K⁺ channels were recorded in insideout patches excised from cultured neonatal rat cardiocytes at 19 °C and at 9 °C. By studying the inhibitory effects of tetraethylammonium (TEA), quinidine and verapamil, the properties of this novel type of K⁺ channel were further characterized. Internal TEA (50 mmol/1) evoked a reversible decline of i_unit to 62.7 + 2.7% of control (at –7 mV), without significant changes of open state kinetics, indicating a blockade of the open K⁺ pore with kinetics too fast to be resolvable at 1 kHz. This TEA blockade was e-fold voltage-dependent, with a decrease of the apparent K_{D( TEA)} from 102 mmol/1 at –37 mV to 65 mmol/1 at +33 mV and, furthermore, became accentuated on lowering the internal K⁺ concentration. Thus, TEA competes with the permeant K⁺ for a site located in some distance from the cytoplasmic margin, within the K⁺ pore. Quinidine (100 mol/l), like verapamil (40 mol/1) reversibly depressed i_unit to about 80% of the control value (at –7 mV), but drug-induced fast flicker blockade proved voltage-insensitive between –27 mV and +23 mV These drugs gain access to a portion of the pore distinct from the TEA binding site whose occupancy by drugs likewise blocks K⁺ permeation. Both drugs showed a greater potency to depress P_o which, with quinidine,decreased reversibly to38.6 ± 11.1% (at –7 mV) and, with verapamil to 24.9 ± 9.1%(at –7 mV), mainly by an increase of the prolonged closed state (C,). This alteration of the gating process also includes a sometimes dramatic shortening of the open state. Most probably, cardiac K_{(outw.-rect.)} ⁺K⁺ _outw.-rect. channels possess a second drug-sensitive site whose occupancy by quinidine or verapamil may directly or allosterically stabilize their non-conducting configuration. Correspondence to: M. Kohlhardt     

Properties of the Ca-activated K channel in pancreatic β-cells

The existence of [Ca²⁺]_i-activated K⁺-channels in the pancreatic β-cell membrane is based in two observations: quinine inhibits K⁺-permeability and, increasing intracellular Ca²⁺ stimulates it. The changes in K⁺-permeability of the β-cell have been monitored electrically by combining measurements of the dependence of the membrane potential on external K⁺ concentration and input resistance. The changes in the passive ⁴²K and ⁸⁶Rb efflux from the whole islet have been measured directly. Intracellular Ca²⁺ has been increased by various means, including increasing extracellular Ca²⁺, addition of the Ca²⁺-ionophore A23187 or noradrenaline and application of mitochondrial uncouplers and blockers. In addition to quinine, many other substances have been found to inhibit or modulate the [Ca²⁺]_i-activated K⁺-channel. The most important of these is the natural stimulus for insulin secretion, glucose. Glucose may inhibit K⁺-permeability by lowering intracellular Ca²⁺. Glibenclamide, a hypoglycaemic sulphonylurea, is about 25 times more active than quinine in blocking the K⁺-channel in β-cells. The methylxanthines, c-AMP, various calmodulin inhibitors and Ba²⁺ also inhibit K⁺-permeability. Genetically diabetic mice have been studied and show an alteration in the [Ca²⁺]_i-activated K⁺-channel.It is concluded that the [Ca²⁺]_i-activated K⁺-channel plays a major role in the normal function of the pancreatic β-cell. The study of its properties should prove valuable for the understanding and treatment of diabetes.     

A computational study of barium blockades in the KcsA potassium channel based on multi-ion potential of mean force calculations and free energy perturbation

Electrophysiological studies have established that the permeation of Ba²⁺ ions through the KcsA K⁺-channel is impeded by the presence of K⁺ ions in the external solution, while no effect is observed for external Na⁺ ions. This Ba²⁺ “lock-in” effect suggests that at least one of the external binding sites of the KcsA channel is thermodynamically selective for K⁺. We used molecular dynamics simulations to interpret these lock-in experiments in the context of the crystallographic structure of KcsA. Assuming that the Ba²⁺ is bound in site S₂ in the dominant blocked state, we examine the conditions that could impede its translocation and cause the observed “lock-in” effect. Although the binding of a K⁺ ion to site S₁ when site S₂ is occupied by Ba²⁺ is prohibitively high in energy (>10 kcal/mol), binding to site S₀ appears to be more plausible (ΔG > 4 kcal/mol). The 2D potential of mean force (PMF) for the simultaneous translocation of Ba²⁺ from site S₂ to site S₁ and of a K⁺ ion on the extracellular side shows a barrier that is consistent with the concept of external lock-in. The barrier opposing the movement of Ba²⁺ is very high when a cation is in site S₀, and considerably smaller when the site is unoccupied. Furthermore, free energy perturbation calculations show that site S₀ is selective for K⁺ by 1.8 kcal/mol when S₂ is occupied by Ba²⁺. However, the same site S₀ is nonselective when site S₂ is occupied by K⁺, which shows that the presence of Ba²⁺ affects the selectivity of the pore. A theoretical framework within classical rate theory is presented to incorporate the concentration dependence of the external ions on the lock-in effect.     

Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca²⁺ and K⁺ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca²⁺ fluorophore and ⁴⁵Ca²⁺-based assays, we demonstrate directly that Letm1 is a Ca²⁺ transporter, with apparent affinities of cations in the sequence of Ca²⁺ ≈ Mn²⁺ > Gd³⁺ ≈ La³⁺ > Sr²⁺ >> Ba²⁺, Mg²⁺, K⁺, Na⁺. Kinetic analysis yields a Letm1 turnover rate of 2 Ca²⁺/s and a K_m of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca²⁺/2 H⁺ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na⁺/Ca²⁺ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H⁺-dependent Ca²⁺ transport mechanism identified in intact mitochondria.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K⁺) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb⁺ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG⁺), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K⁺ (2.66 Å in diameter). However, in the absence of K⁺, significant NMDG⁺ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg²⁺ (1 mM), which blocked the outward NMDG⁺ current, resulting in a strong inward rectification. The NMDG⁺ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG⁺ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K⁺ added to the external NMDG⁺ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K⁺ over NMDG⁺ (P_K⁺/P_NMDG⁺) of ∼240. Reversal potential shifts in mixtures of K⁺ and NMDG⁺ are in accordance with P_K⁺/P_NMDG⁺, indicating that the ions compete for permeation and suggesting that NMDG⁺ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG⁺, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.     

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium,the midgut of tobacco hornoworm (Manduca sexta)

Summary Transbasal electrical potential (V _b) and intraepithelial potassium chemical activity ((K⁺) _i ) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofV _b ranging from +8 to –48 mV (relative to blood side) were recorded. For all sites, (K⁺) _i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than –20 mV show relatively high sensitivity ofV _b to changes in blood side K⁺ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, dye-coupling of several adjacent cells is seen. Low potential sites — those withV _b less negative than –20 mV —typically do not show high sensitivity ofVb to changes of external K⁺, but rather (K⁺) _i rapidly approaches the K⁺ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K⁺) _i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.