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.     

The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies

Although acetylcholine (ACh) is able to activate voltage- and Ca²⁺-sensitive K⁺ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K⁺ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K⁺ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb⁺ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K⁺ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K⁺ current is not carried substantially by BK channels and show that it is carried by a small-conductance K⁺ channel with quite different properties. Received: 28 September 1995/Revised: 26 December 1995     

Cation sensitivity and kinetics of guard-cell potassium channels differ among species

in ward rectifying g uard c ell K ⁺ c hannel, GCKC1_in, from three major crop plants Solanum tuberosum L., Nicotiana tabacum L., and Vicia faba L. Selecting guard cells for our analyses we aimed to test whether K⁺ channels of the same cell type differ among species. The channels shared basic features including voltage-dependence, selectivity and single-channel conductance. They activated at hyperpolarization (V _1/2 ≈ −164 mV) with single channels of 7 pS underlying the whole-cell current. The channel density in S. tuberosum was higher than in V. faba and N. tabacum while the activation and deactivation kinetics were faster in the latter two species. Among different monovalent cations the K⁺ channels discriminated strongly against Na⁺, Li⁺, and Cs⁺. The sensitivity to Cs⁺ was similar for the three species. Extracellular Ca²⁺ blocked the V.␣faba K⁺ channel at concentrations ≥1 mM but only affected its functional homologs in S. tuberosum and N.␣tabacum at higher concentrations and more-negative membrane potentials. Like the differences in Ca²⁺-sensitivity, protoplasts from the three species differed remarkably in their response towards extracellular pH changes. Whereas protons neither altered the open probability nor the kinetic parameters of the V. faba GCKC1_in, in S. tuberosum and N. tabacum this cation affected the voltage-dependent properties strongly. An increase in proton concentration from pH 8.5 to 4.5 shifted the potential of half-maximal open probability to less-negative values with a maximum effect around pH 6.2. The pH modulation of the K⁺ channels could be described assuming a two-state model where the open and closed channel can be protonated. The observed differences in cation-sensitivity and voltage-dependent kinetics between K⁺ channels reflect the diversification of guard-cell channels that may contribute to species-specific variations in the control of stomatal aperture. Received: 19 July 1997 / Accepted: 2 October 1997     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

Two Distinct K+ Channels in Lamprey (Lampetra fluviatilis) Erythrocyte Membrane Characterized by Single Channel Patch Clamp

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K⁺-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 ± 3 pS in isotonic (132 mm K⁺) solutions and 24 ± 2 pS in hypotonic (63 mm K⁺) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K⁺ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg²⁺ and Ca²⁺ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca²⁺ ions on the cytosolic side for activity. Received: 18 July 1997/Revised: 30 January 1998     

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     

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.     

Auxin augments conductance of K+ inward rectifier in maize coleoptile protoplasts

Potassium is taken up by maize (Zea mays L.) coleoptile cells via a typical plant inward rectifier (K _ir ). Sufficient conductance of this channel is essential in order to maintain auxin-stimulated cell elongation. It was therefore investigated whether the activity of this channel is subject to direct or indirect control by this growth hormone. Patch-clamp measurements of whole coleoptile protoplasts revealed no appreciable effect of externally applied 10 μM or 100 μM α-naphthaleneacetic acid (NAA) on the activity of K _ir over test periods of ≥ 18 or ≥ 8 min, respectively. When, however, K _ir was recorded in the cell-attached configiuration and 10 μM NAA administered to the bath medium, the conductance of K _ir increased significantly in 13 out of 18 protoplasts over the control. This rise occurred at a fixed protoplast voltage after a lag period of less than 10 min and exhibited no voltage dependency. The absence of response to NAA of protoplasts in the whole-cell configuration indicates that auxin perception and channel control is linked via a soluble cytoplasmic factor and that this mediator is washed out or modified upon perfusion of the cytoplasm with pipette solution. To search for this expected diffusible factor the K _ir current was recorded before and after elevation of Ca²⁺ and H⁺ in the cytoplasm. In the whole-cell configuration the increase in Ca²⁺ from a nanomolar value to >1 μM by means of Ca²⁺-release from the caged precursor Na₂-DM-nitrophen left K _ir unaffected. The whole-cell K _ir conductance was also not affected upon addition of 10 mM Na⁺-acetate to the bath medium, an operation used to lower the cytoplasmic pH. This excludes a primary role for the known auxin-evoked rise in cytoplasmic Ca²⁺ and H⁺ in K _ir activity. We postulate that another, as yet unknown, mechanism mediates the auxin-evoked stimulation of the number of active K _ir channels in the plasma membrane. Received: 13 May 1998 / Accepted: 9 November 1998     

Characterization of the Driving Force as a Modulator of Gating in Cardiac ATP-sensitive K+ Channels — Evidence for Specific Elementary Properties

Single cardiac ATP-sensitive K⁺ channels and, comparatively, two other members of the inwardly rectifying K⁺ channel family, cardiac K⁺ _(ir) and K⁺ _(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K⁺ gradient for open-state kinetics of these K⁺ channels. The conductive state of K⁺ _(ATP) channels was defined as a function of the electrochemical K⁺ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) τ_open proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K⁺ _(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of τ_open. Since gating continued to operate at E _K , i.e., in the absence of K⁺ permeation through the pore, K⁺ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb⁺ whose interference with an externally located binding site stabilizes the open state. In K⁺ _(ir) and K⁺ _(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K⁺ gradient. Thus, K⁺ _(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus. Received: 24 March 1997/Revised: 24 April 1998     

Pronounced differences between the native K+ channels and KAT1 and KST1 α-subunit homomers of guard cells

Stomatal opening is the result of K⁺-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K⁺-selective ion channels. Here we compare the in-vitro properties of two guard-cell K⁺-channel α-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K⁺-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K⁺-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K⁺ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs⁺, 50% of the guard-cell K⁺-uptake channels (GCKC1_in) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to −90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as −155 mV were required. In contrast, compared to the K⁺ channels in vivo the functional α-subunit homomers almost lacked a voltage-dependent block by extracellular Ca²⁺. Similar to the block by Cs⁺ and Ca²⁺, the acid activation of the α-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCL_in in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K⁺-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K⁺-uptake channels result either from the heterometric assembly of different α-subunits or evolve from cell-type-specific posttranslational modification. Received: 6 March 1998 / Accepted: 9 July 1998     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Reconstitution in Lipid Bilayers of an ATP-Sensitive K+ Channel from Pig Coronary Smooth Muscle

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_ATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na₂. Moreover, it was opened when 10–50 μm 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_ATP 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_ATP channels were characterized by brief openings followed by long closings (open probability, P _o ≈ 0.02); only occasionally (3 out of 12 experiments) did the K_ATP channels have a high open probability (P _o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl₂ to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Current-Voltage Curves of Single CI- Channels which Coexist with Two Types of K+ Channel in the Tonoplast of Chara corallina

A Cl^– channel and two types of K⁺ channel have been observed,by the use of the patch-clamp technique, in the membrane surroundingcytoplasmic droplets from Chara corallina. Measurements on cell-attachedpatches showed that the channel selective for Cl^– hada chord conductance of 21 pS at the resting membrane p.d. (mean= 11 mV, n = 19) and when open, passed an outward current of1.4 pA (n = 24 patches) at the resting p.d., with reversal ofthe direction of current at –54 mV (130 mol m^–3Cl^– in the external solution). The Cl^– concentrationin the cytoplasmic droplet calculated from the reversal p.d.was 15 mol m^–3. The channel strongly rectified outwardcurrent flow, but this rectification disappeared with symmetricalCl^– concentrations across detached patches of membrane.It is concluded that rectification observed in cell-attachedpatches is primarily due to asymmetric ^Cl– concentrationsrather than an asymmetry in energy barriers to Cl^– permeationin the channel or any voltage-dependent kinetics of the channel.The channel was rarely observed in detached patches despitebeing commonly observed in cell-attached patches. However, theabsence of Ca²⁺ at the cytoplasmic face of the membrane allowedobservation of the channel in detached patches for brief periods,during which ion substitution experiments revealed a permeabilitysequence of aspartate (76:33:1). A 100 pS K⁺ channel previously described by Luhring (1986) wasfrequently observed, in some instances simultaneously, witha channel having a conductance of 60 pS and displaying outwardrectification. This rectification was due to the channel remainingopen almost continuously for positive membrane potential differences(p.d.) and remaining shut almost continuously for negative p.d.'s.The 60 pS channel, like the 100 pS K⁺ channel, reversed currentflow at the resting p.d., suggesting that it was also permeableto K⁺. Key words: Plant ion-channels, chloride channel, potassium channel, patch-clamp     

TREK-2 (K2P10.1) and TRESK (K2P18.1) are major background K+ channels in dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K⁺ (K_2P) channels that behave as background K⁺ channels. To identify functional background K⁺ channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K⁺ channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K⁺ channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K_2P channel family. The molecular identity of the three other K⁺ channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K_2P channels, the TRESK-like (14 pS) K⁺ channel was most active at 24°C. At 37°C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K⁺ current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K_2P channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K⁺ conductance of DRG neurons at 37°C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons. two-pore domain K⁺ channel; conductance; excitability     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Electrophysiology of the marine diatom Coscinodiscus wailesii IV: types of non-linear current-voltage-time relationships recorded with single saw-tooth voltage-clamp experiments

Electrophysiological states of the marine diatom Coscinodiscus wailesii are known to change spontaneously in the temporal range of seconds. In order to assess the genuine current-voltage-time relationships of individual states in less than a second, voltage-clamp experiments have been carried out using single sweeps of saw-tooth shaped command voltages. This method is introduced with model calculations. Plotting the results in current-voltage coordinates provides convenient access to several electrophysiological entities, such as absence of drift (smoothly closed IV loops), membrane capacitance (by I jump at sign reversal of dV/dt), and ohmic conductances (in linear regions of the current-voltage relationship), as well as equilibrium voltage (internal intersection of capacitance-corrected, 8-shaped tracings) and coarse gating kinetics (rise or fall of capacitance-corrected I at sign reversal of dV/dt) of a voltage-sensitive ion conductance. From electrophysiological measurements with double-barreled glass-microelectrodes on C. wailesii, several distinct types of current-voltage loops are presented. Most of the data, including recordings from electrical excitation, can be interpreted as temporal relaxations of voltage-sensitive conductances for K⁺ and Cl⁻. A more detailed analysis of the effect of tetraethylammonium (TEA⁺) shows that 10 and 20 mM TEA⁺ inhibit the K⁺ conductance in C. wailesii only by up to about 20% but predominantly via a K⁺ outward rectifier. Received: 23 December 1998 / Revised version: 1 June 1999 / Accepted: 1 June 1999     

Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halophytic angiosperm Zostera muelleri. In whole-cell preparations, both outward and inward rectifying currents that activate in a timeand voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K⁺. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K⁺ than to Na⁺. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K⁺. In addition, the plasma membrane contains a much larger K⁺ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl^– channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd³⁺, an effect that is reversible upon washout.We would like to thank K. Morris and D. McKenzie for technical assistance and the Australian Research Council for financial support.     

Effects of reagents modifying carboxyl groups on the gating current of the myelinated nerve fiber

Summary K⁺ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K⁺-selective channels ranging from 15 to 211 pS in symmetrical 140mm KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140mm KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H⁺ ions (pH=6.8); a 211 pS channel that was Ca²⁺-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917±128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4±8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140mm KCl solutions. Thus, there appears to be a correlation between the presence of functional K⁺ channels and insulin secretion.     

Differential Effect of High Extracellular Ca2+ on K+ and Cl− Conductances in Murine Osteoclasts

Effects of the extracellular Ca²⁺ concentration ([Ca²⁺] _o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca²⁺ was mediated by a Ba²⁺-sensitive, inwardly rectifying K⁺ (IR_K) current. A rise in [Ca²⁺] _o (5–40 mm) inhibited the IR_K current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl⁻ (OR_Cl) current. The activation of the OR_Cl current developed slowly and needed higher [Ca²⁺] _o than that required to inhibit the IR_K current. The inhibition of the IR_K current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the OR_Cl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca²⁺ level neither reduced the IR_K current nor activated the OR_Cl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca²⁺] _o -induced changes in the IR_K and OR_Cl conductances. These results suggest that high [Ca²⁺] _o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IR_K conductance and an activation of an OR_Cl conductance. The two conductances modulated by [Ca²⁺] _o may be involved in different phases of bone resorption because they differed in Ca²⁺ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997