Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

Chloride channels in the luminal membrane of rat pancreatic acini

 Pancreatic acini secrete Na⁺, Cl^–and H₂O in response to secretagogues such as acetylcholine. Cl^–channels in the luminal membrane are a prerequisite for this secretion. The properties of the corresponding conductance have previously been examined using whole-cell recordings. The present study attempts to examine the properties of the single channels in cell-attached and cell-free excised patches from the luminal membrane. To this end the pipettes were filled with an N-methyl-D-glucamine (NMDG⁺) chloride/gluconate solution. The voltage-clamp range was chosen to be pipette positive (cell negative, –60 to –130 mV) in order to increase the driving force for outward Cl^–currents. Under resting conditions cell attached luminal patches had very few single-channel currents (12 out of 45 experiments). Their incidence was sharply increased by carbachol (CCH, 1 μmol/l) in 41 out of 45 experiments. The single-channel conductance of these channels was 1.97 ± 0.05 pS. The properties of these channels in excised patches were examined further: their single-channel conductance was 2.2 ± 0.07 pS (n = 59) and their conductance selectivity was I^– > Br^– > Cl^– >> gluconate. None of the typical Cl^–channel blockers (DIDS, NPPB, glibenclamide 100 μmol/l) blocked these channels. It is concluded that the luminal membrane of the rat pancreatic acinus possesses Cl^–channels with very low conductance which are activated by carbachol. Received: 31 January 1997 / Received after revision: 26 February 1997 / Accepted: 5 March 1997     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

Regulation of ion channels in rat hepatocytes

 Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K⁺ channel with a mean inward conductance of 55 ± 6.5 pS (n = 20) and a mean outward conductance of 25 ± 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl^– channel with a mean outward conductance of 30 ± 4.5 pS (n = 8) and a mean inward conductance of 10 ± 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca²⁺ activity on the intracellular side. Accordingly, the Ca²⁺ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K⁺ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K⁺ channel is inhibited by progesterone, while the outwardly rectifying Cl^– channel is insensitive to progesterone. Received: 21 May 1997 / Received after revision: 7 August 1997 / Accepted: 19 August 1997     

Disruption of actin cytoskeleton attenuates sulfonylurea inhibition of cardiac ATP-sensitive K+ channels

 Two actin filament-depolymerizing agents, DNase I and cytochalasin D, were used to examine the involvement of the cytoskeleton in the functional interaction between the sulfonylurea receptor (SUR) and the ATP-sensitive K⁺ (K_ATP) channels. Isolated rat ventricular cardiomyocytes were studied using open cell-attached patches for single-channel recording. Bath application of DNase I (100 μg/ml) or cytochalasin D (10 μM) stimulated the K_ATP channel activities (in presence of 30 μM ATP), and these channels became resistant to inhibition by tolbutamide (0.5 mM). After exposure to tolbutamide, the relative NP_o value was 0.09 ± 0.02 in control patches in absence of actin disrupters, and 0.67 ± 0.22* or 0.65 ± 0.10*, respectively, in cells treated with DNase I or cytochalasine D (*P < 0.05 vs. control). The inhibitory action of glibenclamide (10 μM) on the K_ATP channels was also attenuated by DNase I. Thus, the disruption of the actin cytoskeleton attenuates the ability of SUR to inhibit the opening of K_ATP channels. Received: 3 February 1997 / Accepted: 25 March 1997     

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging

 In the present work, we have investigated the effects of the aging process on Ca²⁺-activated K⁺ channels (K_Ca2+) of rat skeletal muscle fibres. K_Ca2+ channels of adult (5–7 months old) and aged (24–26 months old) rats were surveyed by the patch-clamp technique. In aged rats, K_Ca2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, K_Ca2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat K_Ca2+ channel, measured in the range of potentials from –60 mV to +20 mV, was about 1.5–2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca²⁺ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5’-O-(3-thiotriphosphate) (ATP[γ-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K⁺ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of K_Ca2+ channels increases with advancing age. Received: 10 April 1997 / Received after revision and accepted: 4 June 1997     

Phosphorylation modulates L-type Ca channels in frog ventricular myocytes by changes in sensitivity to Mg2+ block

 Maitotoxin (MTX) may exert its toxic effect by activating ion conductances and has been shown to elicit a fertilization-like response in Xenopus laevis oocytes. In the present study we investigated the electrophysiological response of stage V–VI Xenopus oocytes to MTX using the two-microelectrode voltage-clamp technique. Membrane voltage (V _m) measurements demonstrated that MTX (50 pM to 1 nM) depolarized the oocytes from –49±7 to –14±1 mV. Subsequent replacement of bath Na⁺ by the impermeant cation NMDG (N-methyl-d-glucamine) shifted V _m from –14±1 to –53±5 mV (n=29). This indicates that MTX activates a cation conductance. Indeed, current measurements at a holding potential of –60 or –100 mV showed that within 10 s of MTX application an inward current component developed which was largely abolished by extracellular Na⁺ removal. After a 1-min application of 1 nM MTX the NMDG-sensitive current increased more than 100-fold from 0.14±0.03 μA to a peak value of 21±3 μA (n=11). The effect of MTX was concentration dependent with an EC₅₀ of about 250 pM but only slowly reversible. Ion substitution experiments indicated that the stimulated conductance was nonselective for monovalent cations with a slight preference for NH₄ ⁺ (2.1) > K⁺ (1.5) > Na⁺ (1.0) > Li⁺ (0.7). Regarding divalent cations, a complex biphasic response to extracellular Na⁺ replacement by Ca²⁺ was observed, which suggests that the stimulated channels may have a small Ca²⁺ permeability but that exposure to high extracellular Ca²⁺ enhances recovery from MTX stimulation. No significant conductance for Mn²⁺ was observed. Application of 1 mM benzamil, 1 mM amiloride, or 100 μM 1-(β-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) reduced the MTX-stimulated inward current by 81%, 62%, or 65%, respectively. Gd³⁺ had an inhibitory effect of 29% and 38% at concentrations of 10 μM or 100 μM, respectively. Flufenamic acid, niflumic acid, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-γ1)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)-ethyl]-acetamide (LOE908), and 3′,5′-dichlorodiphenylamine-2-carboxylic acid (DCDPC), known blockers of other nonselective cation channels, had no significant effect. We conclude that MTX activates a nonselective cation conductance in Xenopus oocytes. The underlying channels may be involved in changes in V _m that occur during the early stages of fertilization. Received: 30 December 1997 / Received after revision and accepted: 17 March 1998     

The role of cytosolic Ca2+ in the secretion of NaCl in isolated in vitro perfused rectal gland tubules of Squalus acanthias

 In many exocrine glands cytosolic Ca²⁺ ([Ca²⁺]_i) plays a pivotal role in stimulation-secretion coupling. In the rectal gland of the dogfish Squalus acanthias this appears not to be the case and it is believed that secretion is mainly controlled by the Cl^– conductance of the luminal membrane. We have examined this question in a study of isolated in vitro perfused rectal gland tubules (RGT). Three types of measurements were performed: (1) measurements of [Ca²⁺]_i by the fura-2 technique; (2) measurements of transepithelial electrical parameters, i.e. transepithelial voltage (V _te), transepithelial resistance (R _te), the equivalent short-circuit current (I _sc) and the voltage across the basolateral membrane (V _bl), and (3) whole-cell patch-clamp measurements of cellular voltage (V _m), conductance (G _m) and membrane capacitance (C _m). The data indicates that carbachol (CCH) increases [Ca²⁺]_i by increasing store release and Ca²⁺ influx. Other agonists, producing cytosolic cAMP, also increased [Ca²⁺] by enhancing Ca²⁺ influx. CCH hyperpolarized these cells and enhanced G _m significantly. The effect of CCH on V _te and I _sc was most marked under control conditions and disappeared in RGT otherwise stimulated by agonists that lead to cAMP production. It is concluded that [Ca²⁺]_i plays a major role in the stimulation of NaCl secretion in RGT by enhancing the basolateral K⁺ conductance. cAMP-producing agonists enhance [Ca²⁺]_i by increased Ca²⁺ influx. CCH releases Ca²⁺ from respective stores. CCH, unlike the cAMP-producing agonists, only increases basolateral K⁺ conductance. It modulates secretion especially under conditions in which the cAMP pathway is not fully activated. Received: 25 November 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Effects of the carcinogen dimethylhydrazine (DMH) on the function of rat colonic crypts

 Rats injected with dimethylhydrazine for 5 weeks (DMH, 40 mg/kg body weight) invariably develop colonic cancer after a latency of some 10–14 weeks. Preliminary studies have suggested that Na⁺ absorption by surface colonic crypt cells is attenuated in the preneoplastic period (8–12 weeks after the first injection of DMH). The present study of glucocorticoid-treated (dexamethasone 6 mg/kg body weight, s.c. 3 days or triamcinolone 30 mg/kg body weight, s.c. 3 days) rats was undertaken to examine the ion transport properties of rat distal colon during this period in more detail. Ussing chamber studies of the distal colon and whole-cell patch-clamp measurements in surface cells, mid-crypt cells and crypt-base cells obtained from isolated crypts were performed. In Ussing chamber studies the equivalent short-circuit current inhibitable by amiloride (10 μmol/l) DMH-treated rats was about 40% of control. In addition, the hyperpolarizing effect of amiloride (10 μmol/l) on membrane voltage (V _m) was strongly attenuated in surface and mid-crypt cells of DMH-treated rats. Carbachol (CCH, 100 μmol/l), which predictably hyperpolarized surface, mid-crypt cells and crypt-base cells of control rats, had no significant effect on V _m in DMH-treated rats, but increased membrane conductance (G _m) significantly. This indicates that CCH probably activates both Cl^–and K⁺ channels in all three colonic crypt compartments in the DMH-treated rats. Forskolin (5 μmol/l), which has the most pronounced effect in crypt-base cells in control rats, depolarized V _m and enhanced G _m in all three compartments in DMH-treated rats. These data indicate that DMH profoundly alters Na⁺ and Cl^–transport in colonic crypts prior to the appearance of colonic adenocarcinoma and that these effects can be summarized as follows: (1) the Na⁺ conductance of surface cells is attenuated; (2) cells along the length of the crypt-lumen axis tend to lose their normal response to CCH and instead show simultaneous and comparable increases in K⁺and Cl^–conductances; (3) the effect of forskolin is enhanced along the entire crypt axis. As a result colonic crypt transport is shifted to predominant Cl^–secretion, findings which are characteristic of colonic carcinoma cell lines such as HT₂₉ and T₈₄ cells. Received: 13 May 1996 / Received after revision: 5 August 1996 / Accepted: 14 October 1996     

Aromatic residues affecting permeation and gating in dSlo BK channels

 Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site. Received: 16 September 1997 / Received after revision: 20 November 1997 / Accepted: 21 November 1997     

Blockade of HERG channels expressed in Xenopus oocytes by external H+

 We have investigated the effect of external H⁺ concentration ([H⁺]_o)on the human-ether-a-go-go-related gene (HERG) current (I _HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I _Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H⁺]_o was increased, the amplitude of the I _HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH_o) values (the potential required for half-maximum activation, V _1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pH_o 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g _max) was also affected by [H⁺]_o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH_o from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca²⁺ concentration, which is also known to block HERG channels. When the extracellular Ca²⁺ concentration was increased from 0.5 mM to 5 mM, the shift in V _1/2 caused by increasing [H⁺]_o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g _max caused by increasing [H⁺]_o was not significantly affected by changing external Ca²⁺ levels. The results indicate that HERG channels are inhibited by [H⁺]_o by two different mechanisms: voltage-dependent blockade (shift of V _1/2) and the decrease in g _max. With respect to the voltage-dependent blockade, the interaction between H⁺ and Ca²⁺ is competitive, whereas for the decreasing g _max, their interaction is non-competitive. Received: 12 January 1999 / Received after revision: 15 February 1999 / Accepted: 16 February 1999     

Intracellular Ca2+ distribution in migrating transformed renal epithelial cells

 Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel. We tested whether a gradient of intracellular Ca²⁺ concentration ([Ca²⁺]_i) underlies the horizontal polarization of K⁺ channel activity. [Ca²⁺]_i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca²⁺]_i indicated that a horizontal gradient exists, with [Ca²⁺]_i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca²⁺]_i in the cell body were found to be 135 ± 34 and 405 ± 59 nmol/l, respectively, whereas they were 79 ± 18 and 307 ± 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K⁺ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca²⁺]_i to test whether its maintenance is due to an uneven distribution of Ca²⁺ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La³⁺ alone or together with charybdotoxin, a specific blocker of Ca²⁺-sensitive K⁺ channels, slowed migration to 47 ± 10% and 9 ± 5% of control, respectively. Local blockade of Ca²⁺ influx into the cell body and the lamellipodium with La³⁺ was followed by a decrease of [Ca²⁺]_i at both cell poles. This points to Ca²⁺ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn²⁺ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca²⁺]_i. We hypothesize that the polarized distribution of intracellular Ca²⁺ stores contributes significantly to the generation of a gradient of [Ca²⁺]_i. Received: 22 July 1996 / Received after revision: 17 December 1996 / Accepted: 10 January 1997     

cAMP Stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance

 Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl^–channel in a large variety of cells expressing this protein. Recently evidence has accumulated that it also regulates other ion channels. A coordinated increase in Cl^–and K⁺ conductances is necessary in many Cl^–-secreting epithelia. This has, for example, recently been demonstrated for the colonic crypt, for which a new type of K⁺ channel and a specific inhibitor of this channel, the chromanol 293B, have been described. In the present study we have examined whether the cAMP-evoked activation of CFTR, overexpressed in Xenopus oocytes, in addition to its known activation of a Cl^–conductance, also upregulates endogenous K⁺ channels. It is shown that CFTR-cRNA-injected but not water-injected oocytes possess a cAMP-activated Cl^–conductance. Of the cAMP-induced whole-cell current increase, 15–25% was due to a 293B-, Ba²⁺and TEA⁺-inhibitable K⁺ conductance. The cRNA of the mutated CFTR (ΔF508 CFTR) had no such effect. We conclude that cAMP activated CFTR and an endogenous IsK-type and 293B-sensitive K⁺ conductance. Similar events, occurring, for example, in the colonic crypt possessing CFTR and 293B-sensitive K⁺ channels, might explain the coordinated cAMP-mediated increase in Cl^–and K⁺ conductances. Received: 12 March 1996 / Accepted: 10 April 1996     

TRESK-like potassium channels in leukemic T cells

In this study, we present patch-clamp characterization of the background potassium current in human lymphoma (Jurkat cells), generated by voltage-independent 16 pS channels with a high ( approximately 100-fold) K(+)/Na(+) selectivity. Depending on the background K(+) channels density, from few per cell up to approximately 1 open channel per mum(2), resting membrane potential was in the range of -40 to -83 mV, approaching E (K) = -88 mV. The background K(+) channels were insensitive to margotoxin (3 nM), apamine (3 nM), and clotrimazole (1 muM), high-affinity blockers of the lymphocyte Kv1.3, SKCa2, and IKCa1 channels. The current depended weakly on external pH. Arachidonic acid (20 muM) and Hg(2+) (0.3-10 muM) suppressed background K(+) current in Jurkat cells by 75-90%. Background K(+) current was weakly sensitive to TEA(+) (IC(50) = 14 mM), and was efficiently suppressed by externally applied bupivacaine (IC(50) = 5 muM), quinine (IC(50) = 16 muM), and Ba(2+) (2 mM). Our data, in particular strong inhibition by mercuric ions, suggest that background K(+) currents expressed in Jurkat cells are mediated by TWIK-related spinal cord K(+) (TRESK) channels belonging to the double-pore domain K(+) channel family. The presence of human TRESK in the membrane protein fraction was confirmed by Western blot analysis.     

The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells

 Previous studies have indicated that a 16-pS K⁺ channel (KC_ca) in the basolateral membrane is responsible for the acetylcholine-induced whole-cell K⁺ conductance in these cells. In the present study we have examined this channel in excised inside-out patches of the basolateral membrane. Over a wide voltage range this channel showed inward rectification. The Ca²⁺ sensitivity was very marked, with a Hill coefficient of three and with half-maximal activation at 330 nmol/l. After several minutes most channels showed a slow run-down. Channel activity could be refreshed by addition of ATP (1 mmol/l) to the bath solution. The non-metabolizable derivative 5’-adenylylimidodiphosphate (AMP-PNP) had no such effect. In contrast, it inhibited channel activity by some 50%. ATP and its derivatives had no effect on the Ca²⁺ sensitivity. Channels activated by ATP were subsequently studied in the presence of alkaline (10 kU/l) or acidic (1 kU/l) phosphatase. Both phosphatases reduced channel activity significantly. These data suggest that the 16-pS K⁺ channel is directly controlled by cytosolic Ca²⁺. This regulatory step is probably distal to an activation produced by protein-kinase-C-dependent phosphorylation. As is the case for several other K⁺ channels, high concentrations of non-metabolizable ATP analogues inhibit this channel. Received: 23 July 1997 / Accepted: 17 September 1997     

视网膜微血管内皮细胞离子通道的特性研究

目的 探讨视网膜微血管内皮细胞离子通道特性。方法 膜片钳技术的全细胞记录方式。结果 原代培养牛视网膜微血管内皮细胞的静息膜电位为(-34.711±2.196)mV(n=46),几乎每次记录都发现相似的跨膜外向电流,该电流具有电压依赖性及不失活特性,能被已知的Ca~(2+)激活K~+通道特异性阻抑剂不同程度阻断。结论 在视网膜微血管内皮细胞上存在Ca~(2+)激活K~+通道。     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Sodium-sensitive, calcium-independent potassium conductance in the leech giant glial cell

 We have measured membrane current, membrane potential and intracellular Na⁺ and Ca²⁺ concentrations, [Na⁺]_i and [Ca²⁺]_i, of the giant glial cell in the nervous system of the leech Hirudo medicinalis using conventional microelectrodes and the fluorescent dyes sodium-binding benzofuran isophthalate (SBFI) and fura-2. When the Na⁺ was removed from the saline, the membrane conductance increased twofold from 1.29±0.1 μS to 2.57±0.18 μS (mean ± SEM; n=27). The rise in membrane conductance was accompanied by a current, which reversed around –74 mV, and the amplitude of K⁺-induced depolarizations or currents increased during Na⁺ removal, suggesting an increase in the K⁺ conductance of the glial membrane. We also monitored [Ca²⁺]_i when removing external Na⁺ in the presence and absence of external Ca²⁺, and during injection of the Ca²⁺-chelator BAPTA into the cells. Our results indicate that Na⁺ modulates a K⁺ conductance of these glial cells, independent of intra- and extracellular Ca²⁺. Received: 1 April 1998 / Received after revision and accepted: 22 May 1998     

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells

(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at –70 mV, and decreased with hyperpolarization and depolarization from –70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to –100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997