Ionic channels in plant cell membranes

The use of patch clamp methods for identifying ion-specific channels and other transport structures in plant cell membranes is described. Methodology, basic concepts that underlie data analysis, and applications of this powerful technique are emphasized.     

Spontaneous inactivation of the Ca-sensitive K channels of human red cells at high intracellular Ca activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a (dCa²⁺/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about −65 mV was observed. At a membrane potential of about −70 mV and an intracellular concentration of about 2·10⁻⁴M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Calcium- and voltage-activated potassium channels in adrenocortical cell membranes

Current flowing through single Ca- and voltage-activated K channels has been recorded from cell-attached and inside-out excised membrane patches of cultured Y-1 adrenocortical cells. In intact cells, single-channel current amplitude and the time a channel stays in the open state increase with membrane depolarization. In excised patches bathed in symmetrical 130 mM K solutions, single-channel conductance is 170 pS. This value is constant in the membrane potential range of +/- 50 mV but decreases at larger hyper- and depolarizations. Channel open probability is heavily influenced by the concentration of ionic Ca at the inner surface of the membrane in the range between 0.01 and 10 microM. When internal Ca concentration is close to 0.01 microM, channels are usually closed even at large depolarizing voltages. With larger Ca concentrations, channel open probability increases and its voltage dependence is greater. These channels are uniformly distributed in the plasma membrane, since one to four channels were seen in more than 99% of the patches isolated in this study. There are previous reports suggesting a role for calcium ions in the secretory response of adrenocortical cells to ACTH. Therefore, it is possible that, as in other endocrine cells, these K channels modulate Ca influx across the plasma membrane and thus contribute to regulate steroid biosynthesis and release.     

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells

Simultaneous measurements of Ca content and ⁴²K⁺ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K⁺ permeability.It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K⁺-permeability mechanism. This demonstrates the existence of a Ca²⁺-refractory state of the K⁺ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.     

Evidence for water channels in renal proximal tubule cell membranes

Summary Water transport mechanisms in rabbit proximal convoluted cell membranes were examined by measurement of: (1) osmotic (P _f ) and diffusional (P _d ) water permeabilities, (2) inhibition ofP _f by mercurials, and (3) activation energies (E _a ) forP _f .P _f was measured in PCT brush border (BBMV) and basolateral membrane (BLMV) vesicles, and in viable PCT cells by stopped-flow light scattering;P _d was measured in PCT cells by proton NMR T_i relaxation times using Mn as a paramagnetic quencher. In BLMV,P _f (0.019 cm/sec, 23°C) was inhibited 65% by 5mm pCMBS and 75% by 300 m HgCl₂ (K _l =42 m);E _a increased from 3.6 to 7.6 kcal/mole (15–40°C) with 300 m HgCl₂. In BBMV,P _f (0.073 cm/sec, 23°C,E _a =2.8 kcal/mole, <33°C and 13.7 kcal/mole, >33°C) was inhibited 65% with HgCl₂ withE _a =9.4 kcal/mole (15–45°C). Mercurial inhibition in BLMV and BBMV was reversed with 10 m mercaptoethanol. Viable PCT cells were isolated from renal cortex by Dounce homogenization and differential seiving. Impedence sizing studies show that PCT cells are perfect osmometers (100–1000 mOsm). Assuming a cell surface-to-volume ratio of 25,000 cm^–1,P _f was 0.010±0.002 cm/sec (37°C) andP _d was 0.0032 cm/sec.P _f was independent of osmotic gradient size (25–1000 mOsm) withE _a 2.5 kcal/mole (<27°C) and 12.7 kcal/mole (>27°C). CellP _f was inhibited 53% by 300 m HgCl₂ (23°C) withE _a 6.2 kcal/mole. These findings indicate that cellP _f is not restricted by extracellular or cytoplasmic unstirred layers and that cellP _f is not flow-dependent. The high BLMV and BBMVP _f , inhibition by HgCl₂, lowE _a which increases with inhibition, and the measuredP _f /P _d >1 in cells in the absence of unstirred layers provide strong evidence for the existence of water channels in proximal tubule brush border and basolateral membranes. These channels are similar to those found in erythrocytes and are likely required for rapid PCT transcellular water flow.     

Water channels in membranes

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (E_a,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had P_d values around 5·10^?3 cm/s at 25 °C, 7 · 10^?3 cm/s at 37 °C with E_a,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the P_d values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of E_a,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylmide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.     

IS3 peptide-formed ion channels in rat skeletal muscle cell membranes

A 22-mer peptide, identical to the primary sequence of domain I segment 3 (IS3) of rat brain sodium channel I, was synthesized. With the patch clamp cell-attached technique, single channel currents could be recorded from the patches of cultured rat myotube membranes when the patches were held at hyperpolarized potentials and the electrode solution contained NaCl and 1 microM IS3, indicating that IS3 incorporated into the membranes and formed ion channels. The single channel conductances of IS3 channels were distributed heterogeneously, but mainly in the range of 10-25 pS. There was a tendency that the mean open time and open probability of IS3 channels increased and the mean close time decreased with the increasing of hyperpolarized membrane potentials. IS3 channels are highly selective for Na+ and Li+ but not for Cl- and K+, similar to the authentic Na+ channels.     

Inhibition of Ca2+-dependent K+ channels by lead in one-step inside-out vesicles from human red cell membranes

Pb2+ modified the apparent threshold sensitivity to Ca2+ of individual K+ channels with a biphasic time-course. At first, the sensitivity to Ca2+ was lowered with the result of a decrease of the fraction of activated vesicles at a given Ca2+ concentration. Later, Pb2+ increased the sensitivity to Ca2+ and the fraction of activated vesicles. The increase of Pb2+ concentration increased the extent of the initial inhibition but decreased its duration. The inhibitory effect was not observed when the addition of Ca2+ preceded the addition of Pb2+. The presence of Mg2+ in the incubation medium was also required. In the absence of Mg2+, Pb2+ decreased the rate of uptake of 86Rb, but no decrease in the fraction of activated vesicles could be demonstrated.     

Analysis of the all or nothing behaviour of Ca-dependent K channels in one-step inside-out vesicles from human red cell membranes

The all or nothing behaviour of Ca2+-dependent K+ channels has been analyzed in one-step inside-out vesicles. There is a threshold for Ca2+ below which the K+ channels remain silent, and which ranges between the 10(-6) and 10(-8) M for different vesicles under the experimental conditions tested, in the absence of Mg2+. The increase of Ca2+ concentration within this range recruits a larger fraction of the vesicles to the active (permeable to 86Rb+) state. The apparent rate of 86Rb+ transport through each individual channel was found to increase, however, with Ca2+ concentration. This finding is not an artefact due to size heterogeneity of the vesicle population, and it is consistent with the variations of the mean open time of the channels with Ca2+ concentration reported previously in patch-clamp experiments. The electron donor system ascorbate + phenazine-methosulphate increases the rate of 86Rb+ transport through the channels whereas oxidized cytochrome c has the opposite effect.     

Physiology and pharmacology of the Na and K channels of axon membranes

The electrical activity of axons is due to changes in Na and K membrane permeabilities. Ions cross the membrane through specific pathways (Na et K channels). Ionic channels are transmembrane proteins forming pores whose openings are controlled by the membrane potential. Their properties are mainly studied by electrophysiological (current recording under voltage clamp conditions) and biochemical (characterization of toxin binding sites) techniques. The purpose of these studies is to determine the structure of these channels and to delineated their mode of functioning.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Hyaluronan export through plasma membranes depends on concurrent K+ efflux by K(ir) channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K(+) channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K(+) channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.     

Transient outward K+ channels in vesicles derived from frog skeletal muscle plasma membranes.

Whole-cell voltage-clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes. Capacitance measurements showed that these vesicles lack invaginations. In solutions containing K+, transient outward currents with reversal potentials close to EK were recorded with a maximum potassium conductance of 0.3 mS/cm2. These currents inactivated in a voltage-dependent manner with a time constant of decay that reached a limiting value of 26 ms at large depolarizations. The steady-state inactivation reached half-maximum values at -66 mV. Transient currents were completely blocked with 5 mM 4-aminopyridine. Single-channel recordings made in inside-out excised patches from the vesicles had ensemble averages with characteristics similar to those of the macroscopic currents, although with significantly faster inactivation time constants. The single-channel chord conductance was 21 pS when the pipette and bath solutions contained 2.5 mM and 120 mM KCl, respectively. It is concluded that these vesicles contain potassium channels that are very similar to A channels found in neurons and other cells.     

Potassium-chloride promiscuous channels in mitochondrial membranes

Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels.     

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chloride channels of intracellular membranes

Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins.