Phosphorylation of K+ channels in the squid giant axon. A mechanistic analysis

Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In squid giant axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.     

Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte

The permeation properties of the 147-pS Ca2+-activated K+ channel of the taenia coli myocytes are similar to those of the delayed rectifier channel in other excitable membranes. It has a selectivity sequence of K+ 1.0 greater than Rb+ 0.65 greater than NH4+ 0.50. Na+, Cs+, Li+, and TEA+ (tetraethylammonium) are impermeant. Internal Na+ blocks K+ channel in a strongly voltage-dependent manner with an equivalent valence (zd) of 1.20. Blockade by internal Cs+ and TEA+ is less voltage dependent, with d of 0.61 and 0.13, and half-blockage concentrations of 88 and 31 mM, respectively. External TEA+ is about 100 times more effective in blocking the K+ channel. All these findings suggest that the 147-pS Ca2+-activated K+ channel in the taenia myocytes, which functions physiologically like the delayed rectifier, is the single-channel basis of the repolarizing current in an action potential.     

Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells

We have cloned a cDNA coding for a delayed rectifier K+ channel from rat brain (RCK1) and rat muscle (RMK1) and expressed it in Xenopus oocytes and in a myoblast cell line (Sol-8). Stably transfected Sol-8 cells exhibited large outward K+ currents, which were indistinguishable from the K+ currents induced in Xenopus oocytes by injection of mRNA transcribed in vitro. RCK1 encodes a K+ channel with a unitary conductance of approximately 14 pS. The steep voltage dependence of channel opening resides in transitions between closed states, whereas the direct transitions into and out of the open state are very rapid and not markedly voltage-dependent. Channel inactivation is very slow, voltage-independent, and occurs from the open state only. We present a simple model that incorporates our findings and is consistent with the presumed structural symmetry of a functional K+ channel.     

External [K+] and the block of the K+ inward rectifier by external Cs+ in frog skeletal muscle.

Frog skeletal muscle has a K+ channel called the inward rectifier, which passes inward current more readily than outward current. Gay and Stanfield (1977) described a voltage-dependent block of inward K+ currents through the inward rectifier by external Cs+ in frog muscle. Here, frog single muscle fibers were voltage clamped using the vaseline-gap voltage-clamp technique to study the effect of external [K+] on the voltage-dependent block of inward K+ currents through the inward rectifier by external Cs+. The block of inward K+ currents through the channel by external Cs+ was found to depend on external [K+], such that increasing the external concentration of the permeant ion K+ potentiated the block produced by the impermeant external Cs+. These findings are not consistent with a one-ion channel model for the inward rectifier. The Eyring rate theory formalism for channels, viewed as single-file multi-ion pores (Hille and Schwarz, 1978), was used to develop a two-site multi-ion model for the inward rectifier. This model successfully reproduced the experimentally observed potentiation of the Cs+ block of the channel by external K+, thus lending further support to the view of the inward rectifier as a multi-ion channel.     

大鼠胰腺β细胞离子通道的一些特性

实验以单个Ｗｉｓｔａｒ大鼠胰腺β细胞为对象,用穿孔膜片箝和细胞贴附式记录技术研究ＡＴＰ敏感Ｋ＾＋通道（ＫＡＴＰ）、延迟整流型Ｋ＾＋通道（ＫＤＲ）、Ｃａ＾２＋通道和Ｎａ＾＋通道的有关特性。结果表明：⑴ＫＡＴＰ通道的内流电导约６５ｐＳ,外流电导约３１ｐＳ,反转电位在－６０ｍＶ左右;⑵ＫＤＲ通道在延迟２０ｍｓ后达到最大激活,ＫＤＲ电流约为ＫＡＴＰ的１／３;⑶钙电流在０ｍＶ左右达到４０－６０ｐＡ的峰值,Ｌ     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Surface charge and calcium channel saturation in bullfrog sympathetic neurons

Currents carried by Ba2+ through calcium channels were recorded in the whole-cell configuration in isolated frog sympathetic neurons. The effect of surface charge on the apparent saturation of the channel with Ba2+ was examined by varying [Ba2+]o and ionic strength. The current increased with [Ba2+]o, and the I-V relation and the activation curve shifted to more positive voltages. The shift of activation could be described by Gouy-Chapman theory, with a surface charge density of 1 e- /140 A2, calculated from the Grahame equation. Changes in ionic strength (replacing N-methyl-D-glucamine with sucrose) shifted the activation curve as expected for a surface charge density of 1 e-/85 A2, in reasonable agreement with the value from changing [Ba2+]o. The instantaneous I-V for fully activated channels also changed with ionic strength, which could be described either by a low surface charge density (less than 1 e-/1,500 A2), or by block by NMG with Kd approximately 300 mM (assuming no surface charge). We conclude that the channel permeation mechanism sees much less surface charge than the gating mechanism. The peak inward current saturated with an apparent Kd = 11.6 mM for Ba2+, while the instantaneous I-V saturated with an apparent Kd = 23.5 mM at 0 mV. This discrepancy can be explained by a lower surface charge near the pore, compared to the voltage sensor. After correction for a surface charge near the pore of 1 e-/1,500 A2, the instantaneous I-V saturated as a function of local [Ba2+]o, with Kd = 65 mM. These results suggest that the channel pore does bind Ba2+ in a saturable manner, but the current-[Ba2+]o relationship may be significantly affected by surface charge.     

Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation

Single-channel K+ currents were recorded from young and mature spinal neurons cultured from Xenopus embryos to examine the bases of the developmental increases in density and in rate of activation of the macroscopic voltage-dependent delayed rectifier K+ current (IKv). K+ channels of three conductance classes (integral of 80, 30, and 15 pS) are present at both ages, but only the intermediate and small conductance classes are voltage-dependent and thus underlie IKv. The increase in the density of IKv is due to increases in the numbers of intermediate and small channels per cell, but not to changes in their open probabilities. The increase in rate of activation of IKv results from a change in the activation kinetics of the intermediate channel class alone.     

Modification of potassium channel kinetics by amino group reagents.

We have examined the actions of several amino group reagents on delayed rectifier potassium channels in squid giant axons. Three general classes of reagents were used: (1) those that preserved the positive charge of amino groups; (2) those that neutralize the charge; and (3) those that replace the positive with a negative charge. All three types of reagents produced qualitatively similar effects on K channel properties. Trinitrobenzene sulfonic acid (TNBS) neutralizes the peptide terminal amino groups and the epsilon-amino group of lysine groups. TNBS (a) slowed the kinetics of macroscopic ionic currents; (b) increased the size of ionic currents at large positive voltages; (c) shifted the voltage-dependent probability of channel opening to more positive potentials but had no effect on the voltage sensitivity; and (d) altered several properties of K channel gating currents. The actions of TNBS on gating currents suggest the presence of multiple gating current components. These effects are not all coupled, suggesting that several amino groups on the external surface of K channels are important for channel gating. A simple kinetic model that considers the channel to be composed of independent heterologous subunits is consistent with most of the modifications produced by amino group reagents.     

Voltage-dependent stimulation of Na+/K(+)-pump current by external cations: selectivity of different K+ congeners.

Currents generated by the endogenous Na+/K+ pump in the oocytes of Xenopus laevis were determined under voltage-clamp as currents activated by different K+ congeners. The voltage dependence of the pump current reflects voltage-dependent steps in the reaction cycle. The decrease of K(+)-activated pump current at positive potentials has been attributed to voltage-dependent stimulation by the external K+ (Rakowski, Vasilets, LaTona and Schwarz (1991) J. Membr. Biol. 121, 177-187). In Na(+)-free solution, activation of the pump by external cations seems to be the dominating voltage-dependent and rate-determining step in the reaction cycle. Under these conditions, the voltage dependence of apparent Km values for pump activation can be analyzed. The dependence suggests voltage-dependent binding of extracellular cations assuming that an effective charge of about 0.4 of an elementary charge is moved in the electrical field during a step associated with the cation binding. The apparent Km values at 0 mV differ for various cations that stimulate pump activity. The values are in mM: 0.10 for Tl+, 0.63 for K+, 0.71 for Rb+, 9.3 for NH4+, and 12.9 for Cs+. The corresponding apparent affinities follow the same sequence as the cation permeability of the K(+)-selective delayed rectifier channel of nerve cells. The results are compatible with the interpretation that the cations have to pass an ion-selective access channel to reach their binding sites in the pump molecule.     

Binding site in eag voltage sensor accommodates a variety of ions and is accessible in closed channel

In ether-a-go-go K+ channels, voltage-dependent activation is modulated by ion binding to a site located in an extracellular-facing crevice between transmembrane segments S2 and S3 in the voltage sensor. We find that acidic residues D278 in S2 and D327 in S3 are able to coordinate a variety of divalent cations, including Mg2+, Mn2+, and Ni2+, which have qualitatively similar functional effects, but different half-maximal effective concentrations. Our data indicate that ions binding to individual voltage sensors in the tetrameric channel act without cooperativity to modulate activation gating. We have taken advantage of the unique phenotype of Ni2+ in the D274A channel, which contains a mutation of a nonbinding site residue, to demonstrate that ions can access the binding site from the extracellular solution when the voltage sensor is in the resting conformation. Our results are difficult to reconcile with the x-ray structure of the KvAP K+ channel, in which the binding site residues are widely separated, and with the hydrophobic paddle model for voltage-dependent activation, in which the voltage sensor domain, including the S3-S4 loop, is near the cytoplasmic side of the membrane in the closed channel.     

Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade

Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.     

重组人白细胞介素—1β对小鼬有骨髓基质细胞K^＋通道的影响

In the present study, both cell-attached and inside-out mode of patch clamp technique were applied to detect the effect of recombinant human interleukin-1 beta (IL-1 beta) on K+ channels of mice bone marrow stromal cells. A 16.7 pS voltage-dependent K+ channel resembling the delayed rectifier K+ channel in excitable cells was identified and characterized. Under stimulation of IL-1 beta (1000 U/ml), the single channel conductance increased to 26.1 +/- 3.6 pS (P < 0.01). IL-1 beta also markedly increased the open time constant and the open probability, but decreased the closed time constant as compared to the same patches without IL-1 beta. In addition, IL-1 beta induced multi-channel activities in about 80 percent patches by inducing more K+ channels of the same type open. These results suggest that activation of K+ channels plays a role in signal transduction of interleukin-1.     

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.     

Elevation of a potassium current in differentiating human leukemic (HL-60) cells

Human promyelocytic leukemia (HL-60) cells display a novel voltage-dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL-60 cells after exposure to phorbol-12-myristate-13-acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl-. From a holding potential of -80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1-2 X 10(-6) M). These properties appear to differ significantly from the Ca2+-activated K+ channel and the delayed rectifier. The increase of this voltage-activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.     

Characterization of the voltage-dependent properties of a volume- sensitive anion conductance

Outwardly rectified, swelling-activated anion conductances have been described in numerous cell types. The major functional variable observed amongst these conductances is the extent and rate of depolarization-induced inactivation. In general, the conductances can be divided into two broad classes, those that show rapid inactivation in response to strong depolarization and those that show little or no voltage dependence. The swelling-activated anion conductance in rat C6 glioma cells is inactivated nearly completely by membrane depolarization above +90 mV and reactivated by membrane hyperpolarization. The kinetics of inactivation and reactivation are fit by single and double exponentials, respectively. Voltage-dependent behavior is well described by a simple linear kinetic model in which the channel exists in an open or one of three inactivated states. pH- induced changes in voltage-dependent gating suggest that the voltage sensor contains critical basic amino acid residues. Extracellular ATP blocks the channel in a voltage-dependent manner. The block is sensitive to the direction of net Cl- movement and increases open channel noise indicating that ATP interacts with the channel pore. Blockage of the channel with ATP dramatically slows depolarization- induced inactivation.     

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.     

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.