Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl^– for gluconate^– shifted the reversal potential, while Cl^– channel blockers, 4,4-diisothiocyanostibene-2,2-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPS (a poorly hydrolyzable analog of ATP), 2,3-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 M suramin (a P2 receptor antagonist), and was partially blocked by 100 M pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca²⁺ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca²⁺-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.     

黄皮酰胺促钾通道开放

一种新发现的具有促智作用的药物——黄皮酰胺能抑制去甲肾上腺素(NE)或KCl引起的血管平滑肌收缩。本研究旨在应用膜片钳(patch clamp)技术探讨黄皮酰胺对Wistar大鼠尾动脉平滑肌细胞膜钾离子通道的作用。单个平滑肌细胞用酶法分离,以细胞封接方式记录离子通道活动。在细胞池内注入2μM黄皮酰胺后,钾离子通道活动明显增强。用本实验室开发的计算机软件(patch clamp analysis system,Version 1.0)计算分析通道活动的特征参数。     

Kinetics and distribution of voltage-gated Ca,Na and K channels on the somata of rat cerebellar Purkinje cells

Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels.     

Extracellular detection of K+ release during migration of transformed Madin-Darby canine kidney cells

 Madin Darby canine kidney cells transformed by alkaline stress (MDCK-F cells) constitutively migrate at a rate of about 1 μm·min^–1. Migration depends on the intermittent activity of a Ca²⁺-stimulated, 53-pS K⁺ channel (K_Ca channel) that is inhibitable by charybdotoxin. In the present study we examined whether this intermittent K_Ca channel activity results in a significant K⁺ loss across the plasma membrane. K⁺ efflux from MDCK-F cells should result in a transient increase of extracellular K⁺ ([K⁺]_e) in the close vicinity of a migrating cell. However, due to the rapid diffusion of K⁺ ions into the virtually infinite extracellular space, such a transient increase in [K⁺]_e was too small to be detected by conventional K⁺-selective electrodes. Therefore, we developed a ”shielded ion-sensitive microelectrode” (SIM) that limited diffusion to a small compartment, formed by a shielding pipette which surrounded the tip of the K⁺-sensitive microelectrode. The SIM improved the signal to noise ratio by a factor of at least three, thus transient increases of [K⁺]_e in the vicinity of MDCK-F cells became detectable. They occurred at a rate of 1.3 min^–1. The cell releases 40 fmol K⁺ during each burst of intermittent K_Ca channel activity, which corresponds to about 15% of the total cellular K⁺ content. Since transmembrane K⁺ loss must be accompanied by anion loss and therefore leads to a decrease of cell volume, these findings support the hypothesis that intermittent volume changes are a prerequisite for the migration of MDCK-F cells. Received: 15 April 1996 / Received after revision: 18 June 1996 / Accepted: 23 July 1996     

Fast and slow blockades of the inward-rectifier K+ channel by external divalent cations in guinea-pig cardiac myocytes

The present patch-clamp study shows that external Mg²⁺, Ca²⁺ and Sr²⁺ decrease the unit amplitude of inward current through the inward-rectifier K⁺ channel in a concentration-dependent manner. Sr²⁺ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr²⁺ binding site () is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg²⁺ or Ca²⁺ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg²⁺ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs⁺ block (=1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs⁺ and Sr²⁺ bind.     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

Epinephrine activates outward rectifying K channel in Madin-Darby canine kidney cells

Patch-clamp recordings were used to study the epinephrine dependent activation of ion channels in the cell membrane of cultured subconfluent renal epithelial (MDCK) cells. The patch-current was dominated by two populations of K channels. The spontaneously active population of K channels shows an inward rectifying behavior. Addition of epinephrine to the cell exterior, after the patchpipette had been sealed to the cell membrane, increased the open probability of the inward rectifying K channel and shifted the membrane potential in the hyperpolarizing direction. The epinephrine induced hyperpolarization occurs in the range of seconds and is caused by activation of outward-rectifying K channels. The outward-rectifying K channel could not be observed under control conditions. Epinephrine activated channels always appeared in clusters of four to nine channels. Both populations of K channels are modulated in their open probability by cytoplasmic free calcium and voltage.     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na⁺ channel currents. Single Na⁺ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na⁺ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na⁺ channel is directly modulated by phosphorylation process through A-kinase.     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.