Calcium- and voltage-activated potassium channels in adrenocortical cell membranes

Current flowing through single Ca- and voltage-activated K channels has been recorded from cell-attached and inside-out excised membrane patches of cultured Y-1 adrenocortical cells. In intact cells, single-channel current amplitude and the time a channel stays in the open state increase with membrane depolarization. In excised patches bathed in symmetrical 130 mM K solutions, single-channel conductance is 170 pS. This value is constant in the membrane potential range of +/- 50 mV but decreases at larger hyper- and depolarizations. Channel open probability is heavily influenced by the concentration of ionic Ca at the inner surface of the membrane in the range between 0.01 and 10 microM. When internal Ca concentration is close to 0.01 microM, channels are usually closed even at large depolarizing voltages. With larger Ca concentrations, channel open probability increases and its voltage dependence is greater. These channels are uniformly distributed in the plasma membrane, since one to four channels were seen in more than 99% of the patches isolated in this study. There are previous reports suggesting a role for calcium ions in the secretory response of adrenocortical cells to ACTH. Therefore, it is possible that, as in other endocrine cells, these K channels modulate Ca influx across the plasma membrane and thus contribute to regulate steroid biosynthesis and release.     

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.     

Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying K_V channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker K_V channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.     

Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae.

Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na+ or K+ equally well, and Ca2+ to a lesser extent. Its open probability (Po) is voltage-dependent, peaking at about -80 mV (cytoplasm negative), and falling to near zero at +80 mV. Elevated cytoplasmic Ca2+, alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K+ over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca2+, this channel is activated by positive going membrane voltages: mean Po is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg2+ or Na+, block the yeast plasma-membrane K+ channel in a similar but less pronounced manner.     

A slow voltage-activated potassium current in rat vagal neurons.

Potassium currents play a key role in controlling the excitability of neurons. In this paper we describe the properties of a novel voltage-activated potassium current in neurons of the rat dorsal motor nucleus of the vagus (DMV). Intracellular recordings were made from DMV neurons in transverse slices of the medulla. Under voltage clamp, depolarization of these neurons from hyperpolarized membrane potentials (more negative than -80 mV) activated two transient outward currents. One had fast kinetics and had properties similar to A-currents. The other current had an activation threshold of around -95 mV (from a holding potential -110 mV) and inactivated with a time constant of about 3s. It had a reversal potential close to the potassium equilibrium potential. This current was not calcium dependent and was not blocked by 4-aminopyridine (5 mM), catechol (5 mM) or tetraethylammonium (20 mM). It was completely inactivated at the resting membrane potential. This current therefore represents a new type of voltage-activated potassium current. It is suggested that this current might act as a brake to repetitive firing when the neuron is depolarized from membrane potentials negative to the resting potential.     

Calcium-activated potassium channels in human platelets

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.     

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.     

Delayed-rectifying potassium channels in mouse peritoneal macrophages: Pharmacological analysis

The effects of 4-aminopyridine, verapamil, and 4-bromophenacylbromide (4-BPB) on the kinetics of delayed outward-rectifying potassium currents (I _K) were investigated in cultured mouse peritoneal macrophages using a classical whole-cell patch-clamp technique. The outwardI _K was completely blocked by 4-aminopyridine at 1.0 mM concentration. Verapamil at the same concentration also blockedI _K completely. Lower concentration (50 µM) of verapamil demonstrated only partial blocking action, which was almost fully reversible, and markedly increased the rate ofI _K inactivation. The main effect of 4-BPB on the outwardI _K was a significant acceleration ofI _K activation and inactivation kinetics. It is suggested that this modulation results from a direct effect of 4-BPB on potassium channels or relates to the arachidonic acid cascade.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 49–53, January–February, 1994.     

Pore stability and gating in voltage-activated calcium channels

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust "all-or-non" device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.     

Ionic channels and membrane hyperpolarization in human macrophages

Summary Microelectrode impalement of human macrophages evokes a transient hyperpolarizing response (HR) of the membrane potential. This HR was found to be dependent on the extracellular concentration of K⁺ but not on that of Na⁺ or Cl^–. It was not influenced by low temperature (12°C) or by 0.2mm ouabain, but was blocked by 0.2mm quinine or 0.2mm Mg²⁺-EGTA. These findings indicate that the HR in human macrophages is caused by the activation of a K⁺ (Ca²⁺) conductance. Two types of ionic channels were identified in intact cells by use of the patch-clamp technique in the cell-attached-patch configuration, low and high-conductance voltage-dependent K⁺ channels. The low-conductance channels had a mean conductance of 38 pS with Na⁺-saline and 32 pS with K⁺-saline in the pipette. The high-coductance channels had a conductance of 101 and 114 pS with Na⁺- and K⁺-saline in the pipette, respectively. Cell-attached patch measurements made during evocation of an HR by microelectrode penetration showed enhanced channel activity associated with the development of the HR. These channels were also high-conductance channels (171 pS with Na⁺- and 165 pS K⁺-saline in the pipette) and were voltage dependent. They were, however, active at less positive potentials than the high-conductance K⁺ channels seen prior to the microelectrode-evoked HR. It is concluded that the high-conductance voltage-dependent ionic channels active during the HR in human macrophages contribute to the development of the HR.     

Charybdotoxin blocks dendrotoxin-sensitive voltage-activated K channels

Charybdotoxin, a short scorpion venom neurotoxin, which was thought to be specific for the blockade of Ca²⁺-activated K⁺ channels also blocks a class of voltage-sensitive K⁺ channels that are known to be the target of other peptide neurotoxins from snake and bee venoms such as dendrotoxin and MCD peptide. Charybdotoxin also inhibits ¹²⁵-dendrotoxin and ¹²⁵I-MCD peptide binding to their receptors. All these effects are observed with an IC₅₀ of about 30 nM.     

电压依赖性钾通道与人类神经性疾病

电压依赖性钾通道是钾通道超家族中成员最多,最为复杂的亚家族,主要包括Kvα亚单位和辅助亚单位两部分,其中快速失活A型通道和毒蕈碱敏感的M通道已被大量研究,它们广泛分布于神经系统,主要参与各种生理和病理作用,如膜兴奋性的产生,神经递质的释放,神经元细胞的增殖和退化,以及神经网络的信号传递等。目前发现Kv通道亚型或亚单位的突变与学习和记忆的损伤,共济失调,癫痫,神经性耳聋等一些神经性疾病的产生有关。     

Functional expression of ERG1 potassium channels in rat alveolar macrophages

Alveolar macrophages (AMs) play a vital role in lung immunity. The recent studies demonstrated that potassium channels were associated with macrophage functions, such as activation, migration and cytokines secretion. However, less is known regarding the expression and function of ERG channels in AMs. Our study showed that ERG1 channel expressed in rat alveolar macrophage, and the expression level was increased when AMs were stimulated with LPS. Furthermore, blockade of ERG1 channels with E4031 down-regulated the mature of ERG1 protein, inhibited NF-κB translocation into the nucleus, and reduced LPS-stimulated IL-6 and IL-1β secretion. These results imply that ERG1 channels are functionally expressed in rat alveolar macrophages and play an important role in inflammatory response.     

Chloride and potassium channels in U937 human monocytes

Summary Ionic channels in a human monocyte cell line (U937) were studied with the inside-out patch-clamp technique. A Ca²⁺-activated K⁺ channel and three Cl^–-selective channels were observed. The Ca²⁺-activated K⁺ channel had an inward-rectifying current-voltage relationship with slope conductance of 28 pS, and was not dependent on membrane potential. Among the three Cl^– channels, and outward-rectifying 28-pS channel was most frequently observed. The permeability ratio (Cl^–/Na⁺) was 4–5 and CH₃SO ₄ ^– was also permeant. The channel became less active with increasing polarizations in either direction, and was inactive beyond ±120 mV. The channel, observed as bursts, occasionally had rapid events within the bursts, suggesting the presence of another mode of kinetics. Diisothiocyanatostilbene-disulfonic acid (DIDS) blocked the channel reversibly in a dose-dependent manner. The second 328-pS Cl^– channel had a linear currentvoltage relationship and permeability ratio (Cl^–/Na⁺) of 5–6. This channel became less active with increasing polarizations and inactive beyond ±50 mV. DIDS blocked the channel irreversibly. The channel had multiple subconductance states. The third 15-pS Cl^– channel was least frequently observed and least voltage sensitive among the Cl^– channels. Intracellular Ca²⁺ or pH affected none of the three Cl^– channels. All three Cl^– channels had a latent period before being observed, suggesting inhibitory factor(s) presentin situ. Activation of the cells with interferon-, interferon-A or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused no change in the properties on any of the channels.     

Retinoic acid blocks potassium channels in human lymphocytes

Using the whole-cell variation of the patch-clamp technique, we have determined that retinoic acid, an active metabolite of natural vitamin A that possesses potent immunomodulating activity, reduces the K+ current in human T lymphocytes and natural killer cells in a dose-dependent manner: acute treatment with 5 X 10(-5) M caused over a 70% reduction while concentrations less than 1 X 10(-5) M caused less than 30% inhibition. Natural killer activity and T cell mitogenesis was inhibited by RA at concentrations that reduced the K+ conductance and correlated with the ability of a variety of classical ion-channel blockers to inhibit the functional activity of these cells. Thus, the reported inhibitory effects on natural killer activity and T cell mitogenesis by high concentrations of retinoic acid can be explained by its effect on the K channel.