Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Activation of an ATP-dependent K+ conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules

In rabbit proximal convoluted tubules, an ATP-sensitive K⁺ (K_ATP) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na⁺ transport and basolateral K⁺ conductance. This K⁺ conductance is inhibited by taurine. We sought to isolate this K⁺ channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K⁺ conductance, largely inhibited by extracellular Ba²⁺ and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K⁺ current which was Ba²⁺-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K⁺ channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K⁺ current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K⁺ current. The possible involvement of AK in the K_ATP channel’s regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.     

Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele

In an attempt to understand the processes mediating ion transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K⁺; the outwardly rectifying conductance was comparatively less selective for K⁺. Membrane hyperpolarization beyond a threshold of about ?70 mV induced inward currents. When E_K was set negative of this threshold, inward currents activated negative of E_K and no outward currents were observed positive of E_K. Outward currents in the stelar protoplasts activated at potentials positive of ?85 mV. However, when E_K was set positive of ?85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K⁺. Inwardly and outwardly rectifying K⁺ channels were observed in outside-out patches from the plasma membrane of cortical and stelar cells, respectively. Characterization of these channels showed that they were likely to be responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K⁺ channel blockers (TEA⁺, Ba²⁺ and Cs⁺). In addition, Ca²⁺ above 1 mM partially blocked the inward current in a voltage-dependent manner but had little effect on the outward current. It is suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important component of the low-affinity K⁺ uptake mechanism (mechanism II) identified in intact roots. The outwardly rectifying conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot.     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary 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_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ 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 () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ 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.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

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     

Studies of the injection of poly(A) protamine mRNA into Xenopus laevis oocytes

When poly(A)⁺ protamine mRNA from trout testes polysomes was injected into living Xenopus oocytes and the latter labelled with [¹⁴C] or [³H]arginine during subsequent incubation, a highly basic, labelled protein fraction was synthesized and could be extracted with 0.5 M H₂SO₄. In the acid extract, a major polypeptide, indistinguishable from trout protamine by several criteria: polyacrylamide and starch gel electrophoreses, carboxymethylcellulose column chromatography, lack of incorporation of [³H]histidine, and autoradiography of tryptic peptides after two-dimensional paper electrophoresis, could be demonstrated. Since no such protein is found in control oocytes injected with saline, it is concluded that poly(A)⁺ protamine mRNA programs the synthesis of trout protamine within Xenopus oocytes. This confirms our previous reports [1–3] that trout testis poly(A)⁺ protamine mRNA can direct the in vitro synthesis of protamine in Krebs II ascites, rabbit reticulocytes and wheat germ cell-free systems. The protamine synthesized upon injection of poly(A)⁺ protamine mRNA into Xenopus oocytes appears to be partially phosphorylated. Injection of increasing amounts of poly(A)⁺ protamine mRNA led to a linear increase in protamine synthesis. The sensitivity of detection was such that less than 1 ng of poly(A)⁺ protamine mRNA gave a significant response. The translational stability of protamine mRNA appeared to be less than that of globin mRNA.     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

The Impermeant Ion Methylammonium Blocks K+ and NH+ 4 Currents through KAT1 Channel Differently: Evidence for Ion Interaction in Channel Permeation

The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different ions in the external medium. With either K⁺, NH⁺ ₄ or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K⁺ and, to a lesser extent, to NH⁺ ₄ but not to the slightly bigger, methylated analogue of NH⁺ ₄, MA. Substituting NH⁺ ₄ for K⁺ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that channel operation depends on the transported substrate. In mixed solution (50 mm K⁺, 50 mm MA) MA inhibited K⁺ current in a voltage-independent manner. The maximum block did not exceed 50% of the K⁺ current. In contrast, when NH⁺ ₄ was the permeant ion (50 mm NH⁺ ₄, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages. The latter block could be partially overcome by the addition of K⁺ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different affinities for binding sites on their permeation pathway. Received: 6 October 1997/Revised: 28 January 1998     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

PKC and AMPK regulation of Kv1.5 potassium channels

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K⁺ current (I_Kur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.     

Cloning and Function of the Rat Colonic Epithelial K+ Channel KVLQT1

K_VLQT1 (KCNQ1) is a voltage-gated K⁺ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K⁺ channel in the colonic epithelium. We now report cloning and expression of K_VLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K_VLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K_VLQT1 in crypt cells and surface epithelium. Expression of rK_VLQT1 in Xenopus oocytes induced a typical delayed activated K⁺ current, that was further activated by increase of intracellular cAMP but not Ca²⁺ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K⁺ conductance in the colonic mucosal epithelium and inhibited whole cell K⁺ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K_VLQT1 is forming an important component of the basolateral cAMP-activated K⁺ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis. Received: 17 July 2000/Revised: 25 October 2000     

Effects of extracellular calcium and protons on osteoclast potassium currents

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca²⁺ and H⁺. We examined the effects of these cations on two types of K⁺ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca²⁺, pH 7.4), the voltage-gated, outwardly rectifying K⁺ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V _0.5). Increasing [Ca²⁺]_out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca²⁺]_out, respectively. This effect of elevating [Ca²⁺]_out was due to a positive shift of the K⁺ channel voltage activation range. Zn²⁺ or Ni²⁺ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K⁺ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K⁺ current, the potency sequence was Zn²⁺ > Ni²⁺ > Ca²⁺. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K⁺ current, changes in the concentration of extracellular H⁺ or Ca²⁺ did not shift the voltage activation range of the inwardly rectifying K⁺ current. These findings are consistent with Ca²⁺ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K⁺ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council.     

Permeation and Gating of an Inwardly Rectifying Potassium Channel : Evidence for a Variable Energy Well

Permeation, gating, and their interrelationship in an inwardly rectifying potassium (K⁺) channel, ROMK2, were studied using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode. The gating kinetics of ROMK2 were well described by a model having one open and two closed states. One closed state was short lived (∼1 ms) and the other was longer lived (∼40 ms) and less frequent (∼1%). The long closed state was abolished by EDTA, suggesting that it was due to block by divalent cations. These closures exhibit a biphasic voltage dependence, implying that the divalent blockers can permeate the channel. The short closures had a similar biphasic voltage dependence, suggesting that they could be due to block by monovalent, permeating cations. The rate of entering the short closed state varied with the K⁺ concentration and was proportional to current amplitude, suggesting that permeating K⁺ ions may be related to the short closures. To explain the results, we propose a variable intrapore energy well model in which a shallow well may change into a deep one, resulting in a normally permeant K⁺ ion becoming a blocker of its own channel.     

Identification and characterization of a novel bacterial ATP-sensitive K<Superscript>+</Superscript> channel

Five bacterial species that are most likely to have putative prokaryotic inward rectifier K⁺ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K⁺ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K⁺ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K⁺ current with characteristics similar to those of the ATP-sensitive K⁺ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.