Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording

Summary The effects of tetraethylammonium ions on currents through high-conductance voltage- and Ca²⁺-activated K⁺ channels have been studied with the help of patch-clamp single-channel and whole-cell current recording on pig pancreatic acinar cells. In excised outside-out membrane patches TEA (1 to 2 mM) added to the bath solution virtually abolishes unitary current activity except at very positive membrane potentials when unitary currents corresponding to a markedly reduced conductance are observed. TEA in a lower concentration (0.2 mM) markedly reduces the open-state probability and causes some reduction of the single-channel conductance. In inside-out membrane patches bath application of TEA in concentrations up to 2 mM has no effect on single-channel currents. At a higher concentration (10 mM) slight reductions in single-channel conductance occur. In whole-cell current recording experiments TEA (1 to 2 mM) added to the bath solution completely suppresses the outward currents associated with depolarizing voltage jumps to membrane potentials of 0 mV and blocks the major part (70 to 90%) of the outward currents even at very positive membrane potentials (30 to 40 mV). In contrast TEA (2 mM) added to the cell interior (pipette solution) has no effect on the outward K⁺ current. Our results demonstrate that TEA in low concentrations (1 to 2 mM) acts specifically on the outside of the plasma membrane to block current through the high-conductance Ca²⁺- and voltage-activated K⁺ channels     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch–clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca²⁺-activated K⁺-selective channel K_Ca3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca²⁺] will cause mtK_Ca3.1 opening, thus linking inner membrane K⁺ permeability and transmembrane potential to Ca²⁺ signalling.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Small Inward Rectifying K+ Channels in Coleoptiles: Inhibition by External Ca2+ and Function in Cell Elongation

Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K⁺ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca²⁺ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca²⁺-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca²⁺-binding and -release, respectively (rate constants >>10³ sec⁻¹) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10³ sec⁻¹). With 10 mm K⁺ and 1 mm Ca²⁺ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K⁺ uptake required for cell elongation. Extracellular TEA or Ca²⁺, which inhibit the K⁺ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca²⁺ and TEA on both processes and the similar Ca²⁺ concentration required for half maximal inhibition of growth (4.3 mm Ca²⁺) and for conductance (1.2 mm Ca²⁺) suggest that K⁺ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles. Received: 6 June 1995/Revised: 12 September 1995     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

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 _DR, 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 _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ 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 _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK 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 _BK 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 [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. 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. Received: 19 September 1995/Revised: 14 March 1996     

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca²⁺-dependent K⁺ current, (ii) a transient K⁺ current inhibited by 4-AP, (iii) an inwardly rectifying K⁺ current blocked by Ba²⁺ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl^? current, (v) a TTX-sensitive Na⁺ conductance and (vi) a L-type Ca²⁺ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca²⁺ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca²⁺-dependent K⁺ influx against the electrochemical gradient, probably via an ouabain-sensitive Na⁺-K⁺ pump.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

The receptor potential of retinular cells of the blowflyCalliphora: The role of sodium,potassium and calcium ions

Summary Simultaneous membrane potential and membrane resistance measurements and current clamp experiments were performed on the visual sense cells ofCalliphora. The results suggest that the receptor potential is due to two different conductance variations: (i) a conductance increase for both Na⁺- and K⁺-ions upon illumination, and (ii) a light-independent conductance increase for K⁺-ions. Evidence is obtained that in the visual sense cells ofCalliphora the light-independent conductance increase is mediated by the influx of Ca²⁺-ions.The conclusion is that the increase of the intracellular Ca²⁺-ion concentration not only decreases the light-induced conductance, but also increases the light-independent conductance. So the potential of the sense cell is governed by two antagonistic conductances. The functional advantages of such a system are discussed.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Calcium-dependent potassium conductance in the photoresponse of a nudibranch mollusk

• 1.1. The response to light of Hermissenda photoreceptors when recorded intracellularly without interference from synaptic and action potentials consisted of three phases: an early depolarization (ED) followed by hyperpolarization (dip) and subsequent depolarization (tail).
• 2.2. The ED and the dip were associated with increased membrane conductance while decreased membrane conductance was involved with the tail.
• 3.3. The dip reversal potential was − 82.1 ± 5.3 mV and its amplitude varied inversely with the log of [K⁺].
• 4.4. Perfusing with agents which block K⁺ current like 4AP, Quinine, Quinidine or injection of TEA eliminated the dip and its associated increased membrane conductance, thus further supporting the role of K⁺ conductance in producing the dip.
• 5.5. The dip was enhanced by increased [Ca²⁺]_o, reduced by decreased [Ca²⁺]_o and abolished together with its associated increased membrane conductance when perfused with either D600, Cd²⁺, Mg²⁺, Mn²⁺, or Co²⁺, which block transmembrane Ca²⁺ current.
• 6.6. The dip and its associated increased membrane conductance were abolished by intracellular injection of EGTA and enhanced by perfusion with Ruthenium red.
• 7.7. Intracellular injection of Ca²⁺ mimicked the dip: membrane conductance was increased and the cell hyperpolarized.
• 8.8. These results indicate that the increase in intracellular [Ca²⁺] is primarily responsible for the light-induced increase of K⁺ conductance during the dip. The possible source of the Ca²⁺ is, at least in part, extracellular due to activation of an inward Ca²⁺ current.

     

Feedback inhibition of sodium uptake in K+-depolarized toad urinary bladders

Ouabain-blocked toad urinary bladders were maintained in Na⁺-free mucosal solutions, and a depolarizing solution of high K⁺ activity containing only 5 mM Na⁺ on the serosal side. Exposure to mucosal sodium (20 mM activity) evoked a transient amiloride-blockable inward current, which decayed to near zero within one hour. The apical sodium conductance increased in the initial phase of the current decay and decreased in the second phase. The conductance decrease required Ca²⁺ to be present on the serosal side and was more rapid when the mucosal Na⁺ activity was higher. At 20 mM mucosal Na⁺ and 3 mM serosal Ca²⁺ the initial (maximal) rate of inhibition amounted to 20% in 10 min. The conductance decrease could be accelerated by raising the serosal Ca²⁺ activity to 10 mM. The inhibition reversed on lowering the serosal Ca²⁺ to 3 μM and, in addition, the mucosal Na⁺ to zero. Exposure of the mucosal surface to the ionophore nystatin abolished the Ca²⁺ sensitivity of the transcellular conductance, showing that the Ca²⁺-sensitive conductance resides in the apical membrane. The data imply that in the K⁺-depolarized epithelia, cellular Ca²⁺, taken up from the serosal medium by means of a Na⁺-Ca²⁺ antiport, cause feedback inhibition by blockage of apical Na⁺ channels. However, the rate of inhibition is small, such that this regulatory mechanism will have little effect at 1 mM serosal Ca²⁺ and less than 20 mM cellular Na⁺.     

Ionic channels and membrane hyperpolarization in human macrophages

Summary Microelectrode impalement of human macrophages evokes a transient hyperpolarizing response (HR) of the membrane potential. This HR was found to be dependent on the extracellular concentration of K⁺ but not on that of Na⁺ or Cl^–. It was not influenced by low temperature (12°C) or by 0.2mm ouabain, but was blocked by 0.2mm quinine or 0.2mm Mg²⁺-EGTA. These findings indicate that the HR in human macrophages is caused by the activation of a K⁺ (Ca²⁺) conductance. Two types of ionic channels were identified in intact cells by use of the patch-clamp technique in the cell-attached-patch configuration, low and high-conductance voltage-dependent K⁺ channels. The low-conductance channels had a mean conductance of 38 pS with Na⁺-saline and 32 pS with K⁺-saline in the pipette. The high-coductance channels had a conductance of 101 and 114 pS with Na⁺- and K⁺-saline in the pipette, respectively. Cell-attached patch measurements made during evocation of an HR by microelectrode penetration showed enhanced channel activity associated with the development of the HR. These channels were also high-conductance channels (171 pS with Na⁺- and 165 pS K⁺-saline in the pipette) and were voltage dependent. They were, however, active at less positive potentials than the high-conductance K⁺ channels seen prior to the microelectrode-evoked HR. It is concluded that the high-conductance voltage-dependent ionic channels active during the HR in human macrophages contribute to the development of the HR.     

Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca²⁺-activated K⁺ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca²⁺-activated K⁺ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca²⁺ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K _0.5 for Ca²⁺ was raised from 20nm at a potential of 0 mV to 300nm at –40 mV. A decrease in pH at the cytoplasmic face of the K⁺ channel reduced the Ca²⁺ sensitivity dramatically. A loss of the high sensitivity to Ca²⁺ was also observed after incubation with MgCl₂, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca²⁺ sensitivity between studies on K⁺ channels from the same tissue. High affinity inhibition (K _0.5=10nm) by charybdotoxin of the Ca²⁺-activated K⁺ channel from the extracellular face could be lifted by an outward flux of K⁺ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K⁺ channels. The high sensitivity for Ca²⁺ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K⁺ channel in the epithelial cells.Dr. E. Moczydlowsky, Yale University School of Medicine, New Haven, CT, and Dr. Per Stampe, Brandeis University, Waltham, MA, are thanked for introduction to the bilayer technique. Tove Soland is thanked for excellent technical assistance. This work was supported by the Novo Nordisk Foundation, the Carlsberg Foundation, the Danish Medical Research Council, and the Austrian Research Council.     

Ionic processes underlying the decrease in osmotic potential of Chara L-cell fragments in dilute electrolyte solutions

Movements of ions are considered to be governed by the electroneutrality rule. Therefore, a cation moving across the cell membrane into the cell either passively or actively should move together with its counterion, an anion, in equal amounts of charge or in exchange for another cation inside the cell. This means that the net influx of the cation in question should be affected by the permeability of its counterion and/or another cation inside the cell. To examine osmotic and ionic regulation in Chara cells, cell fragments of Chara having a lower osmotic pressure than normal (L-cell fragments) were prepared. The L-cell fragments were individually put into various dilute electrolyte solutions and their osmotic potentials were measured with a turgor balance. Concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^?, NO^?₃. and SO^2?₄. in the external electrolyte solutions in which L-cells had been incubated were also analysed by ion chromatography. The results showed that in 0.5 mM KCL + 0.1 mM CaCl₂ solution, Chara L-cell fragments absorbed K⁺ and Cl^? to maintain electroneutrality and then regained their osmotic potential very rapidly. When the anion was Cl, the cation absorbed at the highest rate was K⁺ On the other hand, when the cation was K, the anion absorbed at the highest rate was Cl, Other ions Ca²⁺, SO^2?₄ and NO^?₃ showed much less permeability than K⁺ and Cl ^?for the Chara plasma membrane. The conclusion from these findings was that due to the constraint of electroneutral transport, the uptake rate of a salt into L-cells is limited by the permeability of the least permeable ion.     

Light Response of a Giant Aplysia Neuron

Illumination of an Aplysia giant neuron evokes a membrane hyperpolarization which is associated with a membrane conductance increase of 15%. The light response is best elicited at 490 nM: the neuron also has an absorption peak at this wavelength. At the resting potential (-50 to -60 mV) illumination evokes an outward current in a voltage-clamped cell. This current reverses sign very close to E_K calculated from direct measurements of internal and external K⁺ activity. Increases in external K⁺ concentration shift the reversal potential of the light-evoked response by the same amount as the change in E_K. Decreases in external Na⁺ or Cl^- do not affect the response. Therefore, the response is attributed to an increase in K⁺ conductance. Pressure injection of Ca²⁺ into this neuron also hyperpolarizes the cell membrane. This effect is also due largely to an increase in K⁺ conductance. The light response after Ca²⁺ injection does not appear to be altered. Pressure injection of EGTA abolished or greatly reduced the light response. The effect was reversible. We suggest that light acts upon a single pigment in this neuron, releasing Ca²⁺ which in turn increases K⁺ conductance, thereby hyperpolarizing the neuronal membrane.     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes

Summary 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 Ca²⁺-sensitive K⁺ channel. Reduction of extracellular Ca²⁺ to <1 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 Ca²⁺-sensitive K⁺ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.