Inhibitory effects of dihydropyridines on macroscopic K+ currents and on the large-conductance Ca2+-activated K+ channel in cultured cerebellar granule cells

In cultured cerebellar granule cells, we examined the effects of dihydropyridines (DHPs) on K⁺ currents, using the whole-cell recording configuration of the patch-clamp technique and on Ca²⁺-activated K⁺ channels (maxi K⁺ channels) using outside-out patches. We found that micromolar concentrations of nicardipine, nifedipine, (+) and (–) BAY K 8644, nitrendipine, nisoldipine and (–) nimodipine block 10–60% of macroscopic K⁺ currents. The most potent of these DHPs was nicardipine and the least potent, (–) BAY K 8644. (+) Nimodipine had no effect on this current. The inhibitory effects of nifedipine and nicardipine were not additive with those of 1 mM tetraethylammonium (TEA). Outside-out recordings of maxi K⁺ channels showed a main conductance of 200 pS (in 77% of the patches) and two subconductance states (in 23% of the patches). Neither nifedipine nor nicardipine affected the main conductance, but decreased the values of the subconductance levels. In 10% of these patches, nicardipine induced a flickering activity of the channel. These findings show that both Ca²⁺ and K⁺ channels have DHP-sensitive sites, suggesting similarity in electrostatic binding properties of these channels. Furthermore, cerebellar granule cells may express different subtypes of maxi K⁺ channels having different sensitivities to DHPs. These drugs may provide new tools for the molecular study of K⁺ channels.     

Beta1-subunits increase surface expression of a large-conductance Ca2+-activated K+ channel isoform

Auxiliary (beta) subunits of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the gating properties of the functional channel complex. Here we show that an avian beta1-subunit also stimulates the trafficking of BK(Ca) channels to the plasma membrane in HEK293T cells and in a native population of developing vertebrate neurons. One C-terminal variant of BK(Ca) alpha-subunits, called the VEDEC isoform after its five last residues, is largely retained in intracellular compartments when it is heterologously expressed in HEK293T cells. A closely related splice variant, called QEERL, shows high levels of constitutive trafficking to the plasma membrane. Co-expression of beta1-subunits with the VEDEC isoform resulted in a large increase in surface BK(Ca) channels as assessed by cell-surface biotinylation assays, whole cell recordings of membrane current, and confocal microscopy in HEK293T cells. Co-expression of beta1-subunits slowed the gating kinetics of BK(Ca) channels, as reported previously. Consistent with this, overexpression of beta1-subunits in a native cell type that expresses intracellular VEDEC channels, embryonic day 9 chick ciliary ganglion neurons, resulted in a significant increase in macroscopic Ca(2+)-activated K(+) current. Both the cytoplasmic N- and C-terminal domains of avian beta1 are able to bind directly to VEDEC and QEERL channels. However, overexpression of the N-terminal domain by itself is sufficient to stimulate trafficking of VEDEC channels to the plasma membrane, whereas overexpression of either the cytoplasmic C-terminal domain or the extracellular loop domain did not affect surface expression of VEDEC.     

Inhibition of the human intermediate-conductance, Ca2+-activated K+ channel by intracellular acidification

The effect of changes in pH on the gating properties of the cloned human intermediate-conductance, Ca2+-activated K+ channel (hIK) was studied using the patch-clamp technique. Multi-channel inside-out recordings of patches from HEK-293 cells stably expressing hIK channels revealed that the channel activity is modulated by changes in intracellular pH (pHi). Changes in extracellular pH (pHo) in the range from pH 6.0 to 8.2 did not affect the hIK whole-cell current. Intracellular acidification gradually decreased the activity of the hIK channel, approaching zero activity at pHi 6.0. Decreasing pHi altered neither the conductance nor the inward rectification of hIK channels. The proton-induced inhibition of the multi-channel hIK patch current could not be counteracted by increasing the cytosolic Ca2+ concentration to 30 microM. The molecular sensory mechanism underlying the proton-induced modulation of hIK gating is at present unknown.     

Inhibition of large-conductance Ca(2+)-activated K(+) channels in hippocampal neurons by toosendanin

The effect of toosendanin, a selective presynaptic blocker and effective antibotulismic agent, on large-conductance Ca(2+)-activated K(+) channels was studied in inside-out patches of pyramidal neurons freshly isolated from the hippocampal CA1 region of the rat. Toosendanin (1 x 10(-6)g/ml approximately 1 x 10(-4)g/ml) was found to inhibit large-conductance Ca(2+)-activated K(+) channels by reducing its open probability significantly in a concentration-dependent manner, although the effective concentration of toosendanin was lower in a symmetrical K(+) (150 mM) solution than under asymmetrical conditions (changing K(+) concentration in pipette solution to 5mM). The action was partially reversible by washing. By decreasing the slow open time constant, toosendanin shortened the open dwell time of large-conductance Ca(2+)-activated K(+) channels in a dose-dependent manner. A dose-dependent reduction of unitary current amplitude of the channel was detected after toosendanin perfusion. On elevating the intracellular free calcium concentration from 1 to 10 microM, a similar effect on large-conductance Ca(2+)-activated K(+) channels by toosendanin was also observed, but its efficacy was diminished.These results show that toosendanin inhibits large-conductance Ca(2+)-activated K(+) channels in hippocampal neurons by reducing the open probability and unitary current amplitude of the channel, and that Ca(2+) interferes with the effect. These data provide an explanation for toosendanin-induced facilitation of neurotransmitter release and the antibotulismic effect of the drug.     

A high-conductance Ca2+-activated K+ channel in cultured human eccrine sweat gland cells

Recordings have been made of a potassium-selective ion channel in primary cultures of cells derived from expiants of human eccrine sweat glands obtained from normal subjects and from subjects suffering from cystic fibrosis. There appears to be no functional difference between potassium channels derived from normal subjects and those from cystic fibrosis subjects. The channel falls into the group generally known as Maxi-K channels, and has a slope conductance, with symmetrical solutions in bath and pipette containing 140 mM K⁺, of 230 pS. It is calcium-activated, pH-sensitive and can be blocked by barium and quinine. The channel appears only very rarely in patches derived from confluent cells (in approximately 1 in 30 patches containing channels), but it is more frequently observed in younger cultures of dividing cells derived from recently explanted (within the previous 48–72 h) glands. It is possible that this channel is normally located on the basolateral membrane of the cell, and is responsible for the calcium-dependent secretory and absorptive events seen in the intact sweat gland.     

Effects of aerobic exercise training on large-conductance Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

Purpose

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels provide a negative feedback that regulates vascular tone in the brain circulation. This study investigated the effects of aerobic exercise on gating properties of BK_Ca channels in rat cerebral artery.

Methods

Rats were subjected to moderate-intensity exercise at low (EX-3d/w) and high (EX-5d/w) training volume on a motor-driven treadmill, and compared with age-matched sedentary animals (SED). Inside–out (I/O) patch clamp recording was performed to measure gating properties of the BK_Ca channel.

Results

Aerobic exercise induced a reduction in heart rate and body weight in both training groups. Exercise increased the channel activity, which was more pronounced in EX-5d/w than that in EX-3d/w group. Kinetic analysis revealed that (1) the contribution of short open states was elevated and the duration of both short and long open states were extended by exercising in EX-3d/w; (2) Ex-3d/w had no significant change on conformation of close states; (3) EX-3d/w increased the mean open time without changing mean closed time; (4) EX-5d/w increased both the contribution and duration of long open states; (5) EX-5d/w increased channel mean open time while decreased mean closed time.

Conclusion

The results suggest that regular aerobic exercise may enhance BK_Ca channel activity in cerebral arterial myocytes by changing its biophysical properties, and the electrical remolding induced by exercise may be training volume-dependent.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

平滑肌BKCa通道在运动改变大鼠胸主动脉舒缩特性中的作用

背景：大电导钙激活钾通道(BK_Ca)是调节细胞兴奋性和血管张力的重要离子通道,有关大动脉平滑肌BK_Ca通道的作用机制鲜有报道。 目的：观察有氧运动对大鼠胸主动脉舒缩特性的影响,并探讨平滑肌BK_Ca通道在其中的作用。 方法：将20只Wistar大鼠随机分为安静对照组和有氧运动组,运动组进行12周跑台运动,坡度0°, 20 m/min, 60 min/d, 5 d/周。之后每组5只大鼠行股动静脉插管术。恢复1 d后,于在体、清醒状态下进行心血管功能监测。另各组5只大鼠,取胸主动脉制备去内皮血管环,进行体外血管收缩特性检测。 结果与结论：①有氧运动后静脉注射去甲肾上腺素引起的升压反应幅度下降。②静脉注射BK_Ca通道阻断剂Iberiotoxin可诱发升压反应,且运动组更显著。③ 120 mmol/L KCl在两组胸主动脉血管环均可诱发收缩,最大张力差异无显著性意义。④去甲肾上腺素(10^-9~10^-5 mol/L)诱发的血管收缩呈浓度依赖性,但运动组的最大张力显著低于安静组。⑤Iberiotoxin (3×10^-8 mol/L)预处理血管后,可增强去甲肾上腺素(10^-5 mol/L)诱发的张力增加,且运动组增加幅度显著大于安静组。⑥BKCa通道开放剂NS1619 (10^-10~10^-6 mol/L)可引起去甲肾上腺素诱发的血管收缩张力下降,且运动组对其敏感性(pD2)增加。提示,有氧运动可诱导大鼠心血管反应性和胸主动脉舒缩特性改变,其中平滑肌BKCa通道起着重要作用。     

Toxin pharmacology of the large-conductance Ca2+-activated K+ channel in the apical membrane of rabbit proximal convoluted tubule in primary culture

The patch-clamp technique was used to study the toxin pharmacology of the large-conductance Ca²⁺-activated K⁺ channel (BKCa) present in the apical membrane of rabbit proximal convoluted tubules (PCT) in primary culture. Experiments were performed with the inside-out configuration. This channel was very selective for K⁺ against Na⁺ and had a conductance of 180 pS with 140 mmol/l in the pipette and the bath. The action of toxins was studied on the extracellular side of the channel by using the pipette perfusion technique. Experimental conditions were 140 mmol/l KCl in the pipette and 140 mmol/l Nad in the bath. Pipette potential was maintained at 0 mV. Perfusion of crude venom from Leiurus quinquestriatus hebraeus inhibited reversibly the open probability (P _o) in a concentration-dependent fashion (IC₅₀=0.8 mg/l; n=3). The following synthetic or purified toxins were tested: synthetic charybdotoxin (ChTX) IC₅₀=7.3×10^–9 M (n=5); iberiotoxin (IbTX) IC₅₀=5.5×10^–7 mol/l (n=3); and kaliotoxin (KTX) IC₅₀=4.8×10^–7 mol/l (n=3). The suppression of the six first N-terminal amino-acids slightly reduced the affinity of ChTX (IC₅₀=1.2×10^–8 mol/l, n=4). Neither Dendroaspis polylepis venom nor purified dendrotoxin modified P _o even at high concentrations (20 mg/l and 10^–6 mol/l respectively). Apamin, which blocked the small-conductance K⁺ channel in cultured PCT, did not act on BKCa. These results indicate that ChTX is the most efficient known toxin against the epithelial BKCa in primary cultures of PCT. In spite of there being considerable homology of sequence between ChTX, IbTX and KTX, ChTX was about 100 times more effective than the others. Truncated ChTX kept a high affinity for this channel and could be used to obtain a labelled probe.     

Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.     

Probing a Ca2+-activated K+ channel with quaternary ammonium ions

A series of quaternary amonium (QA) ions were used to probe the gross architecture of the ion conduction pathway in a Ca²⁺-activated K⁺ channel from rat muscle membrane. The channels were inserted into planar phospholipid membranes and the single channel currents were measured in the presence of the different QA ions. Internally applied monovalent QA ions (e.g. tetramethylammonium and analogues) induced a voltage-dependent blockade with a unique effective valence of the block equal to 0.30, and blocking potency increases as the compound is made more hydrophobic. Blockade is relieved by increasing the K⁺ concentration of the internal or external side of the channel. The effective valence of block is independent of K⁺ concentration. These results suggest that, from the internal side, all monovalent QA ions interact with a site located in the channel conduction system. Divalent QA ions of the type n-alkylbis-,-trimethylammonium (bisQn) applied internally also block the channel in a voltage dependent fashion. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 0.41 to 0.27, it remains constant for bisQ5 to bisQ6 and increases up to 0.54 for bisQ10. This dependence of block with chain length implies that 27% of the voltage drop within the channel occurs over a distance of 1 nm. Externally applied monovalent QA ions also block the channel. The site is specific for tetraethylammonium; increasing or decreasing the side chains in one methylene group decrease potency by about 400-fold. It is concluded that the Ca²⁺-activated K⁺ channel has wide mouths located at each end and that they are different in molecular nature.     

Actin filaments regulate the stretch sensitivity of large-conductance,Ca2+-activated K+ channels in coronary artery smooth muscle cells

Using the inside-out patch-clamp technique, large-conductance Ca2+ -activated K+ channel (BK(Ca)) currents were recorded from coronary artery smooth muscle cells. Cytochalasin D, an actin filament disrupter, increased channel activity ( NP(o), where N is the number of channels and P(o) the open probability), and this increase was reversed by phalloidin, an actin filament stabilizer. NP(o) was also increased by colchicine, a microtubule disrupter, and decreased by taxol, a microtubule stabilizer. With the stepwise increase of negative pressure in the patch pipettes, the activity of BK(Ca) gradually increased: the maximum effect (527% increase in NP(o)) was achieved at -40 cmH(2)O and the half-maximum effect at -25 cmH(2)O. The increase in NP(o) in response to negative pressure was abolished by phalloidin but not by taxol. These results imply that both actin filaments and microtubules inhibit the opening of BK(Ca) in coronary artery smooth muscle cells, but that only actin filaments are involved in the stretch sensitivity of BK(Ca).     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Tedisamil blocks single large-conductance Ca2+-activated K+ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein

Enzymatically dispersed smooth muscle cells of the guinea-pig portal vein were studied by the patch-clamp technique. They were found to have Ca²⁺-dependent K⁺ channels with the typical properties of the BK channel, i.e. a reversal potential at the calculated equilibrium potential for K⁺ ions, a striking voltage dependence, and a conductance of approximately 200 pS ([K⁺]₀ 50 mM, [K⁺]_i 150 mM, positive patch potentials). Tedisamil, a new bradycardic agent with an inhibitory action on K⁺ currents in heart muscle, reduced the open probability of the BK channels concentration-dependently (1–100 M) when applied at the cytosolic side of membrane inside-out patches. At 100 M [Ca²⁺]_i, the IC₅₀ of tedisamil was 13.8 M (¯x, n=5). Tedisamil increased the frequency of channel closures, and reduced the mean duration of openings from 8 ms to < 1 ms, while the mean duration of closures within bursts (1–2 ms) was not altered. Tedisamil did not affect long closures (> 160 ms) between bursts, either. The mean time of residence of tedisamil at the BK channel was estimated to be 1–2ms. Hence, tedisamil, in comparison to the slow blocker Ba²⁺ and the fast blocker tetraethylammonium, holds the position of an intermediate K⁺ channel blocker.Supported by the Deutsche Forschungsgemeinschaft     

Activation of extrasynaptic NMDA receptors induces a PKC-dependent switch in AMPA receptor subtypes in mouse cerebellar stellate cells

The repetitive activation of synaptic glutamate receptors can induce a lasting change in the number or subunit composition of synaptic AMPA receptors (AMPARs). However, NMDA receptors that are present extrasynaptically can also be activated by a burst of presynaptic activity, and thus may be involved in the induction of synaptic plasticity. Here we show that the physiological-like activation of extrasynaptic NMDARs induces a lasting change in the synaptic current, by changing the subunit composition of AMPARs at the parallel fibre-to-cerebellar stellate cell synapse. This extrasynaptic NMDAR-induced switch in synaptic AMPARs from GluR2-lacking (Ca²⁺-permeable) to GluR2-containing (Ca²⁺-impermeable) receptors requires the activation of protein kinase C (PKC). These results indicate that the activation of extrasynaptic NMDARs by glutamate spillover is an important mechanism that detects the pattern of afferent activity and subsequently exerts a remote regulation of AMPAR subtypes at the synapse via a PKC-dependent pathway.     

The GPR55 agonist lysophosphatidylinositol acts as an intracellular messenger and bidirectionally modulates Ca2+-activated large-conductance K+ channels in endothelial cells

Lysophospholipids are known to serve as intra- and extracellular messengers affecting many physiological processes. Lysophosphatidylinositol (LPI), which is produced in endothelial cells, acts as an endogenous agonist of the orphan receptor, G protein-coupled receptor 55 (GPR55). Stimulation of GPR55 by LPI evokes an intracellular Ca(2+) rise in several cell types including endothelial cells. In this study, we investigated additional direct, receptor-independent effects of LPI on endothelial large-conductance Ca(2+) and voltage-gated potassium (BK(Ca)) channels. Electrophysiological experiments in the inside-out configuration revealed that LPI directly affects the BK(Ca) channel gating properties. This effect of LPI strictly depended on the presence of Ca(2+) and was concentration-dependent, reversible, and dual in nature. The modulating effects of LPI on endothelial BK(Ca) channels correlated with their initial open probability (Po): stimulation at low Po (<0.3) and inhibition at high Po levels (>0.3). In the whole-cell configuration, LPI in the pipette facilitated membrane hyperpolarization in response to low (0.1-2?μM) histamine concentrations. In contrast, LPI counteracted membrane hyperpolarization in response to supramaximal cell stimulation with histamine. These results highlight a novel receptor-independent and direct bidirectional modulation of BK(Ca) channels by LPI on endothelial cells. We conclude that LPI via this mechanism serves as an important modulator of endothelial electrical responses to cell stimulation.     

Characterization of the high-conductance Ca2+-activated K+ channel in adult human skeletal muscle

Ca²⁺-activated K⁺ channels of a large conductance (BK_Ca) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BK_Ca conductances of 230 pS for symmetrical and 130 pS for physiological K⁺ distributions. Open probability increased with membrane depolarization and increasing internal [Ca²⁺]. The Hill coefficient was 2.0, indicating that at least two Ca²⁺ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5–20 s, was observed following large depolarizations, when extracellular K⁺ was lowered to physiological values. BK_Ca were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC₅₀ approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC₅₀ approx. 5 g/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC₅₀ <10 nM). Measurements on resealed fibre segments at physiological [K⁺] were in accordance with the single-channel data: only when intracellular [Ca²⁺] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K⁺ conductance with depolarizing voltage steps.