Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

川楝素对K+、Ca2+通道活动及细胞内Ca2+浓度的调控

川楝素是我国学者从驱蛔中药中分离、鉴定的一个三萜化合物,已证明具选择地影响神经递质释放,有效地对抗肉毒中毒,促进细胞分化、凋亡,抑制肿瘤增殖,抑制昆虫发育和取食,影响K 、Ca2 通道活动等多种生物效应.综述了证明川楝素抑制多种K 通道,选择地易化L型Ca2 通道和进而升高胞内Ca 浓度的研究资料,并对川楝素产生这些生物效应的机制进行了讨论.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

Neurotransmitter modulation of small-conductance Ca2+-activated K+ channels by regulation of Ca2+ gating

Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation, and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involvesSK2-associated CK2 and results from a 3-fold reduction in the Ca2+ sensitivity of channel gating. CK2phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and increases neuronal excitability in aCK2-dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.     

Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Comparative effects of H+ and Ca2+ on large-conductance Ca2+- and voltage-gated Slo1 K+ channels

Large-conductance Ca²⁺- and voltage-gated Slo1 BK channels are allosterically activated by depolarization and intracellular ligands such as Ca²⁺. Of the two high-affinity Ca²⁺ sensors present in the channel, the RCK1 sensor also mediates H⁺-dependent activation of the channel. In this study, we examined the comparative mechanisms of the channel activation by Ca²⁺ and H⁺. Steady-state macroscopic conductance-voltage measurements as well as single-channel openings at negative voltages where voltage-sensor activation is negligible showed that at respective saturating concentrations Ca²⁺ is more effective in relative stabilization of the open conformation than H⁺. Calculations using the Debye-Hückel formulation suggest that small structural changes in the RCK1 sensor, on the order of few angstroms, may accompany the H⁺-mediated opening of the channel. While the efficacy of H+ in activation of the channel is less than that of Ca²⁺, H⁺ more effectively accelerates the activation kinetics when examined at the concentrations equipotent on macroscopic voltage-dependent activation. The RCK1 sensor therefore is capable of transducing the nature of the ligand bound and transmits qualitatively different information to the channel's permeation gate.     

Growth stimulatory precipitates of Ca2+ and pyrophosphate

Inorganic pyrophosphate (PPi) forms an insoluble precipitate with calcium in growth medium when its concentration exceeds about 0.1 mM. This PPi precipitate can reproduce the effects of 10% calf serum on all cell processes examined in Balb/c 3T3 cells, including hexose uptake and metabolism to lactate, 3H-uridine, and 3H-choline uptake, and the incorporation of 3H-leucine and 3H-thymidine into trichloroacetic acid (TCA)-insoluble material. Concentrations of PPi insufficient to form a precipitate are without effect on cell metabolism. The precipitates are most effective when prepared with concentrations of PPi just sufficient to result in precipitate formation and become considerably less effective as the PPi concentration increases, even though the quantity of precipitate formed continues to increase with PPi concentration up to 1 mM PPi. Precipitates formed at low PPi concentrations consist largely of Ca2+ (81% of cations), PPi (77% of anions), and Pi (23% of anions). Precipitates formed with higher concentrations of PPi contain proportionately less Ca2+ and Pi and more monovalent cations and PPi. We have distinguished cell surface-bound PPi from intracellular PPi by differential extraction. The quantity of surface-bound PPi increases sharply when the PPi concentration reaches the point of precipitate formation. If the precipitate is prevented from binding to the cell surface by inverting monolayer cultures in precipitate-containing medium, the cells are not stimulated. These findings suggest that the binding of PPi precipitate to the cell surface is involved in the stimulation of cell metabolism by PPi. PPi precipitates do not absorb serum mitogens or inhibitors from the culture medium, nor do they affect the binding of 125I-platelet-derived growth factor to its specific cell-surface receptor, suggesting that PPi precipitates do not act directly through either of these mitogen-receptor systems. In analogy to cell stimulation by epidermal growth factor and by antigens, we suggest that PPi may be active only in the form of a precipitate because multivalent binding of receptors with formation of clusters is required for stimulation. The inhibitory effects of high concentrations of PPi may be due to interference by free PPi with formation of active receptor clusters.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Ca2+- and voltage-dependent K+ conductance in dispersed parathyroid cells

The membrane ionic conductances of dispersed parathyroid cells kept in primary culture were studied using the "whole-cell" and "inside-out excised patch" variants of the patch-clamp technique. The major component of the total current was a voltage-dependent outward K+ current without an appreciable inward current. The amplitude of the K+ current was markedly reduced when free internal Ca2+ was buffered by addition of 10 mM EGTA. Recordings of single-channel current in excised membrane patches revealed the presence of K+ channels with large unitary conductance (200 pS in symmetrical 130 mM K+ solutions) which were also activated by depolarization when internal Ca2+ concentration was about 10(-5)-10(-6) M. At any membrane voltage these channels were closed most of the time at internal Ca2+ concentrations lower than 10(-10) M. These results demonstrate the existence of a Ca2+- and voltage-dependent K+ permeability in parathyroid cells which may participate in the unusual membrane potential changes induced by alterations of external Ca2+ and, possibly, in the regulation of parathormone secretion.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.     

Residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) from cells utilizing both the inward Na(+) gradient and the outward K(+) gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.