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Transepithelial current fluctuations were recorded in Necturus gallbladder, clamped at negative as well as positive potentials up to 64 mV. With NaCl-Ringer's (+10 mm TAP) on both sides a mucosa-negative potential enhanced the relaxation noise component, present at zero potential, and produced peaking in the power spectrum at potentials above −36mV. Concomitantly at these potentials an inductive as well as a capacitive low-frequency feature appeared in the impedance locus. Clamping at positive potentials of 18 mV suppressed the relaxation noise component. At potentials above 51mV the spectral values increased predominantly at low frequencies. In this case the power spectrum showed only a 1/ f noise component. The experiments confirm the previous finding that a K efflux through fluctuating apical K channels exists under normal conditions. With serosal KCl-Ringer's the initial Lorentzian component was enhanced at negative but suppressed at positive potentials. The increase at negative potentials was less pronounced than in experiments with NaCl-Ringer's on both sides, indicating saturation of the fluctuating K current component. With mucosal KCl-Ringer's a negative potential depressed the initial relaxation noise component, whereas it was enhanced at +18 mV clamp potential. In the latter case an additional Lorentzian component became apparent at higher frequencies. At potentials of 36 mV and above the low-frequency Lorentzian disappeared whereas the corner frequency of the high-frequency component increased. The latter experiments demonstrate that the relaxation noise component in Necturus gallbladder consists of two superimposed Lorentzians. As the relaxation times of these two components behave differently under an electrical field, there may exist two different types of K channels. It is demonstrated that peaking in the plateau of power spectra can be explained by frequency-dependent attenuation effects, caused by a polarization impedance. [ABSTRACT FROM AUTHOR]

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 ( γ) 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) of the membrane. Voltage dependent time constants of activation of g, 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>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. [ABSTRACT FROM AUTHOR]

The effects of the divalent cations strontium and magnesium on Shaker K channels expressed in Xenopus oocytes were investigated with a two-electrode voltage-clamp technique. 20 mM of the divalent cation shifted activation (conductance vs. potential), steady-state inactivation and inactivation time constant vs. potential curves 10-11 mV along the potential axis. The results were interpreted in terms of the surface charge theory, and the surface charge density was estimated to be -0.27 e nm-2. A comparison of primary structure data and experimental data from the present and previous studies suggests that the first five residues on the extracellular loop between transmembrane segment 5 and the pore region constitutes the functional surface charges. The results further suggest that the surface charge density plays an important role in controlling the activation voltage range. [ABSTRACT FROM AUTHOR]

A K channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K channel (K) because its activity was diminished by the internal application of 50-250 μ m ATP-Na. Moreover, it was opened when 10-50 μ m pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the K channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, K channels were characterized by brief openings followed by long closings (open probability, P ≈ 0.02); only occasionally (3 out of 12 experiments) did the K channels have a high open probability ( P ≥ 0.7). Channel activity could be increased or rescued by adding 2.5-10 m m UDP-TRIS and 0.5-2 m m MgCl to the internal side of the channel. [ABSTRACT FROM AUTHOR]

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K currents, I , present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K channel ( I ) and a Ca-activated maxi-K channel ( I ). I was a classical inward rectifier, conducting large inward currents negative to E and very small outward currents. I was blocked in a voltage-dependent manner by Cs, Na, and Ba, block increasing with hyperpolarization. Block by Na and Ba was time-dependent, whereas Cs block was too fast to resolve. Rb was sparingly permeant. In cell-attached patches with high [K] in the pipette, the single I channel conductance was ∼30 pS and no outward current could be detected. I channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200-250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na. I was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca]. Macroscopic I was blocked by external TEA with half block at 0.35 m m. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. [ABSTRACT FROM AUTHOR]

cGMP reduced the short-circuit current ( I) when applied to the aqueous surface of isolated rabbit and cat ciliary epithelia. cGMP either stimulated (in the rabbit) or had no effect (in the cat) on I when applied to the stromal surface. Addition of the cGMP-mediated hormone atrial natriuretic peptide (ANP) to the stromal (but not the aqueous) surface, or the nitrovasodilator sodium nitroprusside to the stromal surface, inhibited I across rabbit ciliary epithelium. The response to stromal cGMP was partly mediated by K channels at the stromal surface of the rabbit pigmented epithelial (PE) cells, since the effect was inhibited by stromal Ba, and was unaffected by Cl replacement, by bumetanide, or by DIDS. In contrast, the response to aqueous cGMP was not likely mediated by changing either K or Cl channels, based on transepithelial measurements of rabbit ciliary epithelium and complementary whole-cell patch clamping of cultured human nonpigmented ciliary epithelial (NPE) cells. The possibility of interacting effects between cGMP and cAMP in targeting the Na,K-exchange pump was also considered. Strophanthidin blocked the responses to either aqueous or stromal cGAMP. Applying 10 μ m forskolin to generate endogenous cAMP enhanced the subsequent response to aqueous cGMP by ≈80%. We conclude that cGMP has at least two actions on the ciliary epithelium. The major effect may be to reverse cAMP-mediated inhibition of the NPE Na pumps at the aqueous surface of both rabbit and cat ciliary epithelia. The second effect is likely mediated by increasing K-channel and pump activity of the rabbit PE cells at the stromal surface. [ABSTRACT FROM AUTHOR]

Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (IK) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12-18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of IK also requires cell-cell contacts and the presence of 1.8 mM Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 microM TRH, 10 nM PMA or 200 micrograms/ml DiC8. drugs that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of IK triggered by Ca-dependent contacts can be blocked by 110 microM neomycin, 2.0 microM H7, and 250 nM staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca(2+)-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca(2+)-activated contracts. However, the effects of the chemicals tested on IK differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane. [ABSTRACT FROM AUTHOR]

Elementary K+ currents were recorded at 19 degrees C in inside-out patches from cultured neonatal rat cardiocytes to elucidate the block phenomenology in cardiac ATP-sensitive K+ channels when inhibitory drug molecules, such as the sulfonylurea glibenclamide, the phenylalkylamine verapamil or sulfonamide derivatives (HE 93 and sotalol), are interacting in an attempt to stress the hypothesis of multiple channel-associated drug targets. Similar to their adult relatives, neonatal cardiac K(ATP) channels are characterized by very individual open state kinetics, even in cytoplasmically well-controlled, cell-free conditions; at -7 mV, tau open(1) ranged from 0.7 to 4.9 msec in more than 200 patches and tau open(2) from 10 to 64 msec--an argument for a heterogeneous channel population. Nevertheless, a common response to drugs was observed. Glibenclamide and the other inhibitory molecules caused long-lasting interruptions of channel activity, after cytoplasmic application, as if drug occupancy trapped cardiac K(ATP) channels in a very stable, nonconducting configuration. The resultant NPo depression was strongest with glibenclamide (apparent IC50 13 nmol/liter) and much weaker with verapamil (apparent IC50 9 mumol/liter), HE 93 (apparent IC50 29 mumol/liter) and sotalol (apparent IC50 43 mumol/liter) and may have resulted from the occupancy of a single site with drug-specific affinity or of two sites, the high affinity glibenclamide target and a distinct nonglibenclamide, low affinity target. Changes in open state kinetics, particularly in the transition between the O1 state and the O2 state, are other manifestations of drug occupancy of the channel. Any inhibitory drug molecule reduced the likelihood of attaining the O2 state, consistent with a critical reduction of the forward rate constant governing the O1-O2 transition. But only HE 93 (10 mumol/liter) associated (with an apparent association rate constant of 2.3 x 10(6) mol-1 sec-1) to shorten significantly tau open(2) to 60.6 +/- 6% of the pre-drug value, not the expected result when the entrance in and the exit from the O2 state would be drug-unspecifically influenced. Sotalol found yet another and definitely distinctly located binding site to interfere with K+ permeation; both enantiomers associated with a rate close to 5 x 10(5) mol-1 sec-1 with the open pore thereby flicker-blocking cardiac K(ATP) channels. Clearly, these channels accommodate more than one drug-binding domain. [ABSTRACT FROM AUTHOR]

The ectoderm of the one-day chick embryo generates dorsoventrally oriented short-circuit current ( I) entirely dependent on extracellular sodium. At the dorsal cell membrane, the I was modified reversibly and in a concentration-dependent manner by: amiloride (60% decrease at 1 m m, with 2 apparent ICs: 0.13 and 48 μ m), phlorizin (0.1 m m) or removal of glucose (30% decrease, additive to that of amiloride), SITS (1 m m, 13% decrease). Acidification or alkalinization of the dorsal (but not ventral) superfusate produced, respectively, decrease or increase of I with a pH of 7.64. Ba (0.1-1 m m) from either side of the ectoderm decreased the I by 30%. Anthracene-9-carboxylic acid, furosemide and inducers of cAMP had no effect on electrophysiological properties of the blastoderm. The chick ectoderm is therefore a highly polarized epithelium containing, at the dorsal membrane, the high and low affinity amiloride-sensitive Na channels, Na-glucose cotransporter, K channels and pH sensitivity, and, at the ventral membrane, the Na, K-ATPase and K channels. The Na transport reacts to pH, but lacks the cAMP regulatory system, well known in many epithelia. The active Na transport drives glucose and fluid into the intraembryonic space, across and around the blastoderm which, in the absence of blood circulation, could secure renewal of extracellular fluid and disposal of wastes and thus maintain the cell homeostasis. [ABSTRACT FROM AUTHOR]

Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of -100 mV, the SV channel showed half-maximal inhibition with 20 mM tetraethylammonium (TEA), 7 microM 9-amino-acridine, 6 microM (+)-tubocurarine, 300 nM quinacrine, and 35 microM quinine, respectively. The SV channel is also blocked by charybdotoxin (20 nM at -80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol. 61:687-708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred. [ABSTRACT FROM AUTHOR]

We have used whole-cell patch-clamp techniques to examine the sensitivities of the inwardly and the outwardly rectifying K currents in sheep parotid cells to K channel blockers. Extracellular tetraethylammonium (ID ≈ 200 μmol/liter), quinine (ID ≈ 100 μmol/liter), verapamil (ID ≈ 30 μmol/liter) and charybdotoxin (ID < 0.1 μmol/liter) reduced the outwardly rectifying current but had no effect on the inwardly rectifying current. Quinidine inhibited the outwardly rectifying current (ID ≈ 200 μmol/liter) and, at a concentration of 1 mmol/liter, reduced the inwardly rectifying current by 35%. Extracellular Ba inhibited both the inwardly and outwardly rectifying K currents but the inwardly rectifying K current was more sensitive to it (ID ≈ 1 μmol/liter) than was the outwardly rectifying K current (ID ≈ 2 mmol/liter). Extracellular Cs reduced the inwardly rectifying K current (ID ≈ 100 μmol/liter) without affecting the outwardly rectifying current; 4-aminopyridine (1 or 10 mmol/liter), lidocaine (0.1 or 1 mmol/liter) and flecainide (0.01 or 0.1 mmol/liter) affected neither current. In excised outsideout patches, the addition to the bath of quinine (100 μmol/liter), quinidine (100 μmol/liter), verapamil (100 μmol/liter) or charybdotoxin (100 nmol/liter) inhibited Ca- and voltage-sensitive 250 pS K channels (BK channels), but 4-aminopyridine (1 mmol/ liter) and lidocaine (0.1 mmol/liter) did not. The pattern of blocker sensitivities is thus consistent with the hypothesis that BK channels are responsible for the outwardly rectifying whole-cell current seen in resting sheep parotid cells. [ABSTRACT FROM AUTHOR]

We determined differences in the Ca signalling of K* and Cl conductances required for Regulatory Volume Decrease (RVD) in jejunal villus enterocytes passively swollen (0.5 or 0.95, isotonic) compared with swelling because of the absorption of d-glucose ( d-Glc) or l-Alanine ( l-Ala). Cell volume was measured using electronic cell sizing. In nominally Ca-free medium containing EGTA (100 μ m) RVD after 0.5 or 0.95, isotonic challenge was prevented. l-Ala swelling and subsequent RVD was influenced in Ca-free medium. Villus cells were incubated with 10 μ m of the acetomethoxy derivative of 1,2.bis (2-aminophenoxy) ethane N,N,N,N tetracetic acid (BAPTA-AM) and RVD after 0.5 · isotonic swelling or l-Ala swelling was prevented. Niguldipine (0.1 μ m), nifedipine (5 μ m), diltiazem (100 μ m), Ni, and Co (1 m m) all prevented hypotonic RVD but had no effect on RVD after l-Ala addition. Charybdotoxin (25 n m) a potent inhibitor of Ca-activated K channels, had no effect on hypotonic RVD but prevented RVD of villus cells swollen by d-Glc. We used the calmodulin antagonists, napthalene sulfonamide derivatives W-7 and W-13, to assess calmodulin activation of K and Cl conductance in these two models. l-Ala swelling and subsequent RVD was not influenced by 25 μ m W-7; hypotonic RVD was prevented by 25 μ m W-7 or 100 μ mW-13. The W-13 inhibition of RVD was by-passed with 0.5 μ m gramicidin. Our data show that hypotonic RVD requires extracellular Ca and that the K conductance activated is not charybdotoxin sensitive but requires calmodulin. Na-nutrient RVD requires intracellular calcium mobilization to activate a charybdotoxin-sensitive K conductance. The signalling for RVD after cell swelling because of transport of osmotically active Na nutrient is different than the signalling for RVD after passive hypotonic cell swelling. [ABSTRACT FROM AUTHOR]

The properties of transporters (or channels) for monovalent cations in the membrane of isolated pancreatic zymogen granules were characterized with an assay measuring bulk cation influx driven by a proton diffusion potential. The proton diffusion potential was generated by suspending granules in an isotonic monovalent cation/acetate solution and increasing the proton conductance of the membrane with a protonophore. Monovalent cation conductance had the sequence Rb > K > Na > Cs > Li > N-methyl glucamine. The conductance could be inhibited by Ca, Mg, Ba, and pharmacological agents such as quinine, quinidine, glyburide and tolbutamide, but not by 5 m m tetra-ethyl ammonium or 5m m 4-aminopyridine, when applied to the cytosolic surface of the granule membrane. Over 50% of K conductance could be inhibited by millimolar concentrations of ATP or MgATP. The inhibition by MgATP, but not by ATP itself, was reversed by the K channel opener diazoxide. The inhibitory effect is probably by a noncovalent interaction since it could be mimicked by nonhydrolyzable analogs of ATP and by ADP. The reversal of MgATP inhibition by diazoxide may be mediated by phosphorylation since it was not affected by dilution, and was blocked by the protein kinase inhibitor H7. The properties of the K conductance of pancreatic zymogen granule membranes are similar to those of ATP-sensitive K channels found in the plasma membrane of insulin-secreting islet cells, neurons, muscle, and renal cells. [ABSTRACT FROM AUTHOR]

Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP were blocked by ruthenium red, a blocker of an IP-gated Ca-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l- cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP or cAMP when the buffering capacity for Ca of the pipette solution was increased, when extracellular Ca was removed or after addition of 20-200 n m charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca] that opened Ca-activated K channels. The results suggest that different conductances modulated by either IP or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca-activated K channels contribute to the termination of the IP and cAMP responses. [ABSTRACT FROM AUTHOR]

Membrane K currents of malignant lymphocytes (Nb2 cells) were studied with the whole-cell patch-clamp method. Upon depolarization, K currents activate with a delay and follow a sigmoid time course, resembling other delayed rectifier K currents present in nerve and muscle cells. The activation time constant of these currents is voltage dependent, increasing from 1 msec at +90 mV to approximately 37 msec at −30 mV. The fractional number of open channels has a sigmoid voltage dependence with a midpoint near −25 mV. Deactivation of K currents in Nb2 cells is voltage dependent and follows a simple exponential time course. Time constant of this process increases from 5 msec at −115 mV to almost 80 msec at −40 mV. The relative permeability of K channels to different monovalent cations follows the sequence: K (1) > Rb (0.75) > NH(0.11) > Cs (0.07) > Na (0.05). Inactivation of K currents is a biexponential process with time constants of approximately 600 and 7,000 msec. Inactivation of K currents in Nb2 cells is not a voltage-dependent process. The steady-state inactivation curve of K currents has a midpoint near −40 mV. Following a 500-msec voltage pulse, inactivation of K currents recovers with a simple exponential process with a time constant of 9 sec. Short duration (∼50 msec) voltage-clamp pulses do not induce significant inactivation of these currents. K currents in malignant lymphocytes do not display the phenomenon of cumulative inactivation as described for other delayed rectifier-type K channels. Application of a train of voltage pulses to positive potentials at different frequencies induces a moderate decrease in peak outward currents. The use of substances (N-bromoacetamide, trypsin, chloramine-T, and papain) that remove the inactivation of Na and K currents in other cells are not effective in removing the inactivation of K currents present in this lymphoma cell line. Significant differences were found between the characteristics of K currents in this malignant cell line and those present in normal lymphocytes. Possible physiological implications for these differences and for the role of K currents in the proliferation of normal and malignant lymphocytes are discussed. [ABSTRACT FROM AUTHOR]

Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K conductance. Whole-cell currents showed an N-shaped current-voltage ( I−13; V) relation with inward current activated at potentials negative to E. When external K was varied, I-V curves shifted 53 mV/10-fold change in [K], as predicted for a K-selective channel. Inward current was blocked by Ba and showed a time-dependent decline at negative potentials, which was reduced in Na-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance (γ) was 31 pS when recorded in the cell-attached configuration with 140 m m K in the pipette and was found to be dependent on [K]. (iii) Addition of Ba to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border. [ABSTRACT FROM AUTHOR]

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 m m external K (with 5 m m Ca-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage( V) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean V= −143± 4 mV), the other (mean V= −237± 10 mV) exhibited an appreciable background of primary H transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective Kequilibrium potential ( E), in 3 and 10 m m external K. In these cases Vwas also negative of the activation voltage for the inward rectifying K current, thus creating a strong bias for passive K uptake through inward-rectifying K channels. In contrast, when pump activity was absent Vwas situated positive of Eand cells revealed a bias for K efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 μ m ABA to the medium. These depolarizations activated current through outward-rectifying K channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization. The analyses also yield indirect evidence for a rise in cytoplasmic Ca activity in ABA, as well as for a K current distinct from the dominant inward and outward-rectifying K channels, but neither support nor discount a role for Ca influx in depolarizing the membrane. A striking similarity was found for the modulation of inward currents either in response to ABA or after spontaneous depolarizations. This renders the possibility of an agonist (i.e., ABA) activated Ca current across the plasma membrane as trigger for the voltage transitions unlikely. [ABSTRACT FROM AUTHOR]

We have used the nystatin perforated patch technique to study ionic currents in rat pancreatic β-cells. The access resistance ( R) between the pipette and the cell cytoplasm, measured by analyzing capacitive currents, decreased with a slow exponential time course (τ=5.4±2.7 min) after seal formation. As R decreased, the magnitude of voltage-dependent K and Ca currents increased with a similar time course, and their activation kinetics became faster. After R stabilized, the macroscopic currents remained stable for up to an hour or more. When the final R was sufficiently low, Ca tail currents could be resolved which had properties similar to those recorded with the classical whole-cell technique. Two types of K channels could be characterized with perforated patch recordings of macroscopic K currents: (i) ATP-blockable K (K) channels which generate a time and voltage independent current that is blocked by glyburide and enhanced by pinacidil and (ii) voltage-dependent K (K) channels. Whole-cell recordings of K currents in the absence of ATP in the pipette showed that the maximum K conductance of the β-cell was 83.8±40 nS. Perforated patch recordings show that the resting K conductance is 3.57±2.09 nS, which corresponds to about 4% of the channels being open in the intact β-cell. In classical whole-cell recordings. K activation kinetics become faster during the first 10-15 min of recording, probably due to a dissipating Donnan potential. In perforated patch recordings where the Donnan potential is very small, K activation kinetics were nearly identical to the steady-state whole cell measurements. [ABSTRACT FROM AUTHOR]

The effects of charybdotoxin (CTX) on single [Ca]-activated potassium channel ( K) activity and whole-cell K currents were examined in rat and mouse pancreatic β-cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured β-cells and β-cells in intact islets were compared. K activity was very infrequent at negative patch potentials (−70< V<0 mV), channel activity appearing at highly depolarized V. K open probability at these depolarized V values was insensitive to glucose (10 and 20 mm) and the metabolic uncoupler 2,4 dinitrophenol (DNP). However, DNP blocked glucose-evoked action potential firing and reversed glucose-induced inhibition of the activity of K channels of smaller conductance. The venom from Leiurus quinquestriatus hebreus (LQV) and highly purified CTX inhibited K channel activity when applied to the outer aspect of the excised membrane patch. CTX (5.8 and 18 nm) inhibited channel activity by 50 and 100%, respectively. Whole-cell outward K currents exhibited an early transient component which was blocked by CTX, and a delayed component which was insensitive to the toxin. The individual spikes evoked by glucose, recorded in the perforated-patch modality, were not affected by CTX (20 nm). Moreover, the frequency of slow oscillations in membrane potential, the frequency of action potentials and the rate of repolarization of the action potentials recorded from pancreatic islet β-cells in the presence of glucose were not affected by CTX. We conclude that the K does not participate in the steady-state glucose-induced electrical activity in rodent pancreatic islets. [ABSTRACT FROM AUTHOR]

The effect of lowering intracellular pH on the membrane potential ( E) of rat thymic lymphocytes was studied using the potential-sensitive dye bis-oxonol. Cells were acid loaded by addition of the electroneutral K/H exchanging ionophore nigericin. Acidification to pH 6.3 in Na-free solution resulted in a biphasic change in E: an early transient hyperpolarization followed by a sustained depolarization. These changes were associated with a rise in cytosolic free Ca ([Ca]). The hyperpolarization was eliminated when the change in [Ca] was prevented using BAPTA, an intracellular Ca chelator. Moreover, a similar hyperpolarization was elicited by elevation of [Ca] at physiological pH using ionomycin, suggesting involvement of Ca-activated K channels. In contrast, the depolarization phase could not be mimicked by raising [Ca] with ionomycin. However, intracellular BAPTA effectively inhibited the acidificationinduced depolarization. Inhibition was also obtained by extracellular addition of EGTA or dithiothreitol, even when the external free Ca concentration remained unaltered. These observations suggested a possible role of contaminating trace metals. Cytosolic acidification is envisaged to induce intracellular accumulation of one or more trace metals, which induces the observed changes in E. Accordingly, similar changes in E can be induced without acidification by the addition of small amounts of Cu to the medium. The ionic basis of the E changes induced by acidification and the significance of these observations are discussed. [ABSTRACT FROM AUTHOR]

Patch-clamp and single cell [Ca2+]i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of -62 +/- 2 mV (n = 42) and an average basal [Ca2+]i of 102 +/- 6 nM (n = 37), glucose (2.5-10 mM): (i) depolarized the membrane, through a decrease in the outward KATP current, (ii) evoked Ca2+ spike potentials, and (iii) caused a sharp rise in [Ca2+]i. In the continued presence of glucose both cromakalim (100-200 microM) and diazoxide (100 microM) repolarized the membrane, terminated Ca2+ spike potentials and attenuated the secretagogue-induced rise in [Ca2+]i. In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K+ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K+ channels, reversed the effects of cromakalim on membrane potential and KATP currents. [ABSTRACT FROM AUTHOR]

Human red cells were prepared with various cellular Na and K concentrations at a constant sum of 156 mm. At maximal activation of the K conductance, g(Ca), the net efflux of K was determined as a function of the cellular Na and K concentrations and the membrane potential, V, at a fixed [K] of ∼3.5 mm. V was only varied from ( V− E)≈25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988). g(Ca) as a function of cellular Na and K concentrations at V=−40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na. Since the present Ca-activated K channels have been shown to be of the multi-ion type, the experimental data from each set of Na and K concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101 mm. Data from a previous study of voltage dependence as a function of the degree of Ca activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence of g(Ca) for outward currents at ( V− E)>25 25 mV reflects a voltage-dependent Na block of the Ca-activated K channels. [ABSTRACT FROM AUTHOR]

We have studied a class of Ca-dependent K channels in inside-out excised membrane patches from Paramecium under patch clamp. Single channels had a conductance of 72 ±9.0 pS in a solution containing 100 mM K. The channels were selective for K over Rb with the permeability ratio of 1∶ 0.56. and over Na, Cs or NH with a ratio 1∶<0.1. The channel activity was dependent on Ca, which was applied to the cytoplasmic side; the Ca concentration for the half maximal activation was 2 μ m. The Hill coefficient for the Ca dependence of the channel activity was 2.58, indicating that more than two Ca bindings are necessary for full activation. Unlike most Ca-dependent K channels in other organisms, the channels in Paramecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV per e-fold change in channel activity. While a high Ca concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba and Ca were relatively ineffective in blockage. It was concluded that these Ca-dependent K channels in Paramecium are different from the previously described Ca-dependent K channels, and are perhaps of a novel class. [ABSTRACT FROM AUTHOR]

The patch-clamp technique was used to identify and investigate two K channels in the cell membrane of the HIT cell, an insulin secreting cell line with glucose-sensitive secretion. Channel characteristics were compared with those of glucose-modulated K channels in the RINm5F cell, an insulin secreting cell line in which secretion is largely glucose insensitive. A 65.7 pS channel, identified with the ATP-sensitive K channel was seen in HIT cell-attached patches. Channel activity was dose-dependently inhibited by glucose, by 50 and 100% at 450 μ m and 8 mm glucose, respectively, similar to the values previously reported for RIN cells. In inside-out patches channel activity was 50% inhibited by 56 μ m ATP and completely blocked between 500 μ m and 1 mm, again, similar to the values reported for RIN cells. As in RIN cells a second, considerably larger (184 pS), K channel was glucose sensitive; the glucose sensitivity was similar to that in RIN cells with 50 and 100% channel inhibition at 7.5 and 25 mm, respectively. After patch excision the mean channel conductance increased from 184 to 215 pS. Under these conditions activity was strongly calcium dependent in the range pCa 5-7, identifying this as a calcium- and voltage-dependent K (K(Ca, V)) channel; the calcium sensitivity was similar to that of the adult rat β cell K(Ca, V) channel. In inside-out RIN cell patches, the large K channel was less abundant but displayed a similar conductance (223 pS). However, its calcium sensitivity was more than 10 times lower than in HIT cells, similar to that of the K(Ca, V) channel in the neonatal rat β cell, which also displays a reduced secretory response to glucose. Based on these observations, it is proposed that the low calcium sensitivity of the K(Ca, V) channel may be causally associated with secretory deficiency in RIN cells and the immature secretory response of the neonatal β cell. [ABSTRACT FROM AUTHOR]

The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca-sensitive K channel. Reduction of extracellular Ca to <1 μ m by the addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na/K ATPase, Na/H antiporter and Na/K/Cl cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K by triggering the opening of Ca-sensitive K channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena. [ABSTRACT FROM AUTHOR]

The presence and regional localization of voltagegated ion channels on taste cells in Necturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na, K, or Ca channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (1) voltage-gated K channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na and Ca channels are found on the apical (chemoreceptive) and basolateral (synaptic) membrane; (iii) there is a K leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K conductance may underlie receptor potentials ellicted by rapid stimuli. [ABSTRACT FROM AUTHOR]

Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials >20 mV and an inwardly rectifying K current present at potentials negative to −60 mV. Tail currents elicited by voltage steps that activated outward current reversed near E, indicating that the outward current was due to a K conductance. The I-V curve for the macroscopic outward current was similar to the mean single-channel I-V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in >85% of patches from macrophages cultured for >7 days. Only 35% of the human macrophages cultured for >7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K] increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin, J. Membrane Biol. 103:41-53, 1988). [ABSTRACT FROM AUTHOR]

The outer membrane of mechanically prepared protoplasmic droplets from Acetabularia mediterranea has been investigated by patch-clamp techniques. These membranes are shown to consist of physiologically intact plasmalemma. With the Cl pump inhibited, microscopic currents through K-selective channels were studied. These currents compare well with macroscopic K currents as previously determined by standard microelectrode techniques and tracer flux measurements. There is about one K channel per μm in the plasmalemma. The current-voltage relationship ( I−V curve) of the main open channel (channel A) is sigmoid over a voltage range between about −100 and +100 mV with saturation currents of about ±10 pA. A second species (or different state of channel A) of K-selective channels (channel B) differs from channel A by smaller saturation currents (about ±7 pA) and a much smaller open probability. The open probability of channel A increases from almost zero at large negative voltages to about 1/2 at large positive voltages. Taking the closed times into account, the mean steady-state I-V curve of channel A displays outward rectification about the equilibrium voltage for K and negative slope conductance at larger negative voltages. The open channel I-V curve of the open channels A and B, the changes of the I-V curve of the open channel A upon variation of the external K concentration, as well as the mean steady-state I-V curves of channel A are described by simple reaction kinetic models, the parameters of which are determined to fit the experimental data. The results are discussed with respect to data from other K channels in plants and with respect to regulation of the cytoplasmic K concentration in Acetabularia. [ABSTRACT FROM AUTHOR]

The current-voltage ( I/V) technique was employed to investigate the different electrophysiological states of the Chara plasmalemma and their interaction under a range of conditions. In K state the membrane became very permeable (conductances >20 S m 2) as [K] increased to 10 mm. As the cells were then easily damaged by the voltage-clamp procedures, it was difficult to determine the saturation K conductance. TEA (tetraethylammonium chloride) reversibly blocked the K channels, but had no effect on the I/V curve of the pump state, indicating that the K channels were not participating in this state. Acid pH (4.5) diminished the K conductance, but did not alter the response of the K channels to change in [K]. Alkaline pH (11.0) made Chara resting PD 'bistable': the PD either stayed near the estimated E and the I/V curve showed a negative conductance region typical of the K state, or it hyperpolarized and the near-linear I/V profile of the proton-permeable state was observed. [ABSTRACT FROM AUTHOR]

The potential difference across the membrane of Hydrodictyon africanum was controlled by voltage clamping and positive and negative steps in the PD were applied. For positive steps in the PD to values less negative than a threshold value, there is a PD and time-dependent increase in the outward current which has an S-shaped time course. Following the cessation of these steps, the current reverses instantaneously and declines with a simple time course. These currents show a strong K dependence and are blocked by tetraethylammonium (TEA) and nonyltriethylammonium (C) ions, suggesting that they arise from the opening and then the closing of K channels. There is also a PD and time-dependent increase in the inward currents in response to negative steps in the membrane PD. The membrane properties have been described by three current-voltage curves, for the instantaneous current, for the steady-state current and for the current flow when the K channels are open. The response of the unclamped or free-running membrane PD to steps of constant current can be accounted for by the observed kinetics of the opening and closing of the K channels. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) are capable of both self-renewal and multilineage differentiation; thus, they can be expanded in vivo or in vitro and differentiated to produce different cell types. Despite their biological and medical interest, many physiological properties of undifferentiated mESCs, such as ion channel function, are not fully understood. Ion channels are thought to be involved in cell proliferation and differentiation. The aim of this study was to characterize functional ion channels in cultured undifferentiated mESCs and their role in cell proliferation. L-type voltage-activated Ca channels sensitive to nifedipine and small-conductance Ca-activated K (SK) channels sensitive to apamin were identified. Ca-activated K currents were blocked by millimolar concentrations of tetraethylammonium. The effects of Ca channel and Ca-activated K channel blockers on the proliferation of undifferentiated mESCs were investigated by bromodeoxyuridine (BrdU) incorporation. Dihydropyridine derivatives, such as nifedipine, inhibited cell growth and BrdU incorporation into the cells, whereas apamin, which selectively blocks SK channels, had no effect on cell growth. These results demonstrate that functional voltage-operated Ca channels and Ca-activated K channels are present in undifferentiated mESCs. Moreover, voltage-gated L-type Ca channels, but not SK channels, might be necessary for proliferation of undifferentiated mESCs. [ABSTRACT FROM AUTHOR]

The slack ( slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles. [ABSTRACT FROM AUTHOR]

Potassium channels play important physiological roles in human syncytiotrophoblasts (hSTBs) from placenta, an epithelium responsible for maternal-fetal exchange. Basal and apical plasma membranes differ in their lipid and protein composition, and the latter contains cholesterol-enriched microdomains. In placental tissue, the specific localization of potassium channels is unknown. Previously, we described two isolated subdomains from the apical membrane (MVM and LMVM) and their respective microdomains (lipid rafts). Here, we report on the distribution of K2.1, K2.1, TASK-1, and TREK-1 in hSTB membranes and the lipid rafts that segregate them. Immunoblotting experiments showed that these channels are present mainly in the apical membrane from healthy hSTBs. Apical expression versus basal membrane was 84 and 16% for K2.1 and K2.1, 60 and 30% for TREK-1, and 74 and 26% for TASK-1. Interestingly, K2.1 showed differences between apical membrane subdomains: 26 ± 8% was located in the LMVM and 59 ± 9% in MVM. In pathological placentas, the expression distribution changed in the basal membrane: preeclampsia shifted to 50% and intrauterine growth restriction to 42% for TASK-1 and both pathologies increased to 25% for K2.1 and K2.1, K2.1 appeared to be associated with rafts that were sensitive to cholesterol depletion in healthy, but not in pathological, placentas. K2.1 and TREK-1 emerged in the nonraft fractions. The precise membrane localization of ion channels in hSTB membranes is necessary to understand the physiological events. [ABSTRACT FROM AUTHOR]

Afferent innervation patterns in the vestibular periphery are complex, and vestibular afferents show a large variation in their regularity of firing. Calyx fibers terminate on type I vestibular hair cells and have firing characteristics distinct from the bouton fibers that innervate type II hair cells. Whole-cell patch clamp was used to investigate ionic currents that could influence firing patterns in calyx terminals. Underlying K(Ca) conductances have been described in vestibular ganglion cells, but their presence in afferent terminals has not been investigated previously. Apamin, a selective blocker of SK-type calcium-activated K(+) channels, was tested on calyx afferent terminals isolated from gerbil semicircular canals during postnatal days 1-50. Lowering extracellular calcium or application of apamin (20-500 nM) reduced slowly activating outward currents in voltage clamp. Apamin also reduced the action potential afterhyperpolarization (AHP) in whole-cell current clamp, but only after the first two postnatal weeks. K(+) channel expression increased during the first postnatal month, and SK channels were found to contribute to the AHP, which may in turn influence discharge regularity in calyx vestibular afferents. [ABSTRACT FROM AUTHOR]

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K(+) than the blood and express various membrane and water transporters (Na(+)/K(+)-ATPase; Ca(2+)-ATPase; V-type ATPase; Cl(-) channels; CFTR Cl(-) channels; K(+) channels; L-, T- and N-type Ca(2+) channels; Na(+)/H(+) exchangers; Na(+)-driven HCO(3) (-)/Cl(-) exchangers (NDCBEs); Na(+)/HCO(3) (-) cotransporters (NBCes); Na(+)-K(+)-2Cl(-) cotransporter; Na(+)/Ca(2+) exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions. [ABSTRACT FROM AUTHOR]

KCNE1-KCNE5 are single membrane-spanning proteins that associate with voltage-gated potassium channels to diversify their function. Other than the KCNQ1/KCNE1 complex, little is known about how KCNE proteins work. We focus on KCNE2, which associates with KCNQ1 to form K channels critical for gastric acid secretion in parietal cells. We use cysteine (Cys)-scanning mutagenesis to probe the functional role of residues along the KCNE2 transmembrane domain (TMD) in modulating KCNQ1 function. There is an α-helical periodicity in how Cys substitutions along the KCNE2 TMD perturb KCNQ1 pore conductance/ion selectivity. However, positions where Cys substitutions perturb KCNQ1 gating kinetics cluster to the extracellular end and cytoplasmic half of the KCNE2 TMD. This is the first systematic perturbation analysis of a KCNE TMD. We propose that the KCNE2 TMD adopts an α-helical secondary structure with one face making intimate contact with the KCNQ1 pore domain, while the contacts with the KCNQ1 voltage-sensing domain appear more dynamic. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A proper rate of programmed cell death or apoptosis is required to maintain normal tissue homeostasis. In disease states such as cancer and some forms of hypertension, apoptosis is blocked, resulting in hyperplasia. In neurodegenerative diseases, uncontrolled apoptosis leads to loss of brain tissue. The flow of ions in and out of the cell and its intracellular organelles is becoming increasingly linked to the generation of many of these diseased states. This review focuses on the transport of K(+) across the cell membrane and that of the mitochondria via integral K(+)-permeable channels. We describe the different types of K(+) channels that have been identified, and investigate the roles they play in controlling the different phases of apoptosis: early cell shrinkage, cytochrome c release, caspase activation, and DNA fragmentation. Attention is also given to K(+) channels on the inner mitochondrial membrane, whose activity may underlie anti- or pro-apoptotic mechanisms in neurons and cardiomyocytes. [ABSTRACT FROM AUTHOR]

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel’s voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4’s positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the ‘mutant cycle analysis’ to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538. [ABSTRACT FROM AUTHOR]

We have measured fluid secretion rate in Rhodnius prolixus upper Malpighian tubules (UMT) stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. Thirteen pharmacological agents were applied: ouabain, Bafilomycin A(1), furosemide, bumetanide, DIOA, Probenecid, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter-transporters and ion and water channels. The basic assumption is that water movement changes reflect changes in ion transport mechanisms, which we localize as follows: (i) At the basolateral cell membrane, fundamental are a Na(+)-K(+)-2Cl(-) cotransporter and a Cl(-)-HCO(3) (-) exchanger; of intermediate importance are the Na(+)-K(+)-ATPase, Cl(-) channels and Rp-MIP water channels; K(+) channels play a lesser role: (ii) At the apical cell membrane, most important are a K(+)-Cl(-) cotransport that is being located for the first time, a V-H(+)-ATPase; and a Na(+)-H(+) exchanger; a urate-anion exchanger and K(+) channels are less important, while Cl(-) channels are not important at all. A tentative model for the function of the UMT cell is presented. [ABSTRACT FROM AUTHOR]

We used the patch-clamp technique to identify and characterize the electrophysiological, biophysical, and pharmacological properties of K(+) channels in enzymatically dissociated ventricular cells of the land pulmonate snail Helix. The family of outward K(+) currents started to activate at -30 mV and the activation was faster at more depolarized potentials (time constants: at 0 mV 17.4 +/- 1.2 ms vs. 2.5 +/- 0.1 ms at + 60 mV). The current waveforms were similar to those of the A-type family of voltage-dependent K(+) currents encoded by Kv4.2 in mammals. Inactivation of the current was relatively fast, i.e., 50.2 +/- 1.8% of current was inactivated within 250 ms at + 40 mV. The recovery of K(+) channels from inactivation was relatively slow with a mean time constant of 1.7 +/- 0.2 s. Closer examination of steady-state inactivation kinetics revealed that the voltage dependency of inactivation was U-shaped, exhibiting less inactivation at more depolarized membrane potentials. On the basis of this phenomenon, we suggest that a channel encoded by Kv2.1 similar to that in mammals does exist in land pulmonates of the Helix genus. Outward currents were sensitive to 4-aminopyridine and tetraethylammonium chloride. The last compound was most effective, with an IC(50) of 336 +/- 142 micro mol l(-1). Thus, using distinct pharmacological and biophysical tools we identified different types of voltage-gated K(+) channels. [ABSTRACT FROM AUTHOR]

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K(+) channels. We report here the cloning of a K(+) channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K(+) channel characteristics. The amino-acid sequence of the eel K(+) channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism. [ABSTRACT FROM AUTHOR]

K2P channel is the leaky potassium channel that is critical to keep up the negative resting membrane potential for legitimate electrical conductivity of the excitable tissues. Recently, many substances and medication elements are discovered that could either straightforwardly or in a roundabout way influence the 15 distinctive K + ion channels including TWIK, TREK, TASK, TALK, THIK, and TRESK. Opening and shutting of these channels or any adjustment in their conduct is thought to alter the pathophysiological condition of CNS. There is no document available till now to explain in detail about the molecular mechanism of agents acting on K2P channel. Accordingly, in this review we cover the current research and mechanism of action of these channels, we have also tried to mention the detailed effect of drugs and how the channel behavior changes by focusing on recent advances regarding activation and modulation of ion channels., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We studied the relationship between changes in intracellular pH (pH(i)), intracellular Ca(2+)([Ca(2+)](i)) and charybdotoxin sensitive (CTX) maxi-K(+) channels occurring after modest 'physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH(i) and [Ca(2+)](i) by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 x isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 x isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 x isotonic dilution, the pH(i) alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this DeltapH(i) and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH(4)Cl permitted complete RVD which could be inhibited by CTX. The rate of (86)Rb efflux which also increased after this 0.93 x isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na(+)-free medium. Modest swelling transiently increased [Ca(2+)](i) and Ca(2+)-free medium or blocking alkalinization by MIA or Na(+)-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca(2+)-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca(2+)](i) and volume changes became sensitive to CTX. We conclude that both alkalinization of pH(i) and increased [Ca(2+)](i) observed with 'physiological' volume increase are essential for the activation of CTX-sensitive maxi-K(+) channels required for RVD. [ABSTRACT FROM AUTHOR]

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K(+) efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 microM by 26%, and 50 microM totally. Fluoxetine had a triphasic effect on K(+) efflux; from 20 to 100 microM it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 microM by 25%, and 50 microM completely. Bumetanide (1 microM) inhibited RVI by 43%. In a Cl(-)-free medium, fluoxetine (50-500 microM) progressively inhibited bumetanide-insensitive K(+) influx. The inhibitions of RVI and K(+) influx induced by fluoxetine 20 to 50 microM were similar to those induced by 1 microM bumetanide and by Cl(-)-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K(+) channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K(+) efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 microM inhibits NKCC, and at 100-1000 microM inhibits the Na(+) pump. One possible explanation for these findings is that fluoxetine could interact with K(+)-selective sites in K(+) channels, the NKC cotransporter and the Na(+) pump. [ABSTRACT FROM AUTHOR]

The activation kinetics of outward currents in protoplasts from barley root xylem parenchyma was investigated using the patch-clamp technique. The K(+) outward rectifying conductance (KORC), providing the main pathway for K(+) transport to the xylem, could be described in terms of a Hodgkin-Huxley model with four independent gates. Gating of KORC depended on voltage and the external K(+) concentration. An increase in the external K(+) concentration resulted in a shift in the voltage dependence of gating. This could be explained by a K(+) dependence of the rate constant beta for channel closure, indicating binding of K(+) to a regulatory site exposed to the bath. Occasionally, KORC was observed to inactivate; this inactivation occurred and vanished spontaneously. In some of the whole cell and excised patch recordings, a stepwise increase in outward current was observed upon a depolarizing voltage pulse, indicating that several populations of 'sleepy' channels existed in the plasma membrane that activated with a certain lag time. It is discussed whether this observation can be explained by a putative subunit, which retards channel activation, or by a scheme of cooperative gating. A quantitative description of outward rectifying K(+) channels in xylem parenchyma cells is a major step forward towards a mathematical model of salt transport into the xylem. [ABSTRACT FROM AUTHOR]

The most frequently observed K+ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch-clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimics the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pKa 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca2+ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca2+ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme. [ABSTRACT FROM AUTHOR]

The effect of pressure upon the delayed, K, voltage-clamp currents of giant axons from the squid Loligo vulgaris was studied in axons treated with 300 nm TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, ΔV, of 31 cm/mole at 15°C; ΔV increased to about 42 cm/mole at 5°C. Pressure slightly increased the magnitude, but did not produce any obvious major change in the voltage dependence, of the steady-state K conductance estimated from the current jump at the end of step depolarizations of small amplitude (to membrane potentials, E, ≦20 mV) and relatively short duration. At higher depolarizations, pressure produced a more substantial increase of the late membrane conductance, associated with an apparent enhancement of a slow component of the K conductance which could not be described within the framework of the Hodgkin-Huxley (HH) n kinetic scheme. The apparent ΔV values that characterize the pressure dependence of the early component of the K conductance are very close to those that describe the effect of pressure on Na activation kinetics, and it is conceivable that they are related to activation volumes involved in the isomerization of the normal K channels. The enhancement of the slow component of membrane conductance by pressure implies either a large increase in the conductance of the ionic channels that are responsible for it or a strong relative hastening of their turn-on kinetics. [ABSTRACT FROM AUTHOR]

The Ca-activated maxi K channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na reabsorption from the intestinal lumen. Previous measurements of these basolateral K channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca with a K ranging from 20 n m to 300 n m, whereas other studies have a much lower sensitivity to Ca. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K channels lost their high sensitivity to Ca, yet they could still be activated by high concentrations of Ca (10 μ m). Furthermore, the high sensitivity to Ca could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30-120 sec for dephosphorylation and of 10-280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca of the maxi K channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca sensitivities for maxi K channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. [ABSTRACT FROM AUTHOR]