Molecular insights into the structure and function of plant K(+) transport mechanisms

Our understanding of plant potassium transport has increased in the past decade through the application of molecular biological techniques. In this review, recent work on inward and outward rectifying K(+) channels as well as high affinity K(+) transporters is described. Through the work on inward rectifying K(+) channels, we now have precise details on how the structure of these proteins determines functional characteristics such as ion conduction, pH sensitivity, selectivity and voltage sensing. The physiological function of inward rectifying K(+) channels in plants has been clarified through the analysis of expression patterns and mutational analysis. Two classes of outward rectifying K(+) channels have now been cloned from plants and their initial characterisation is reviewed. The physiological role of one class of outward rectifying K(+) channel has been demonstrated to be involved in long distance transport of K(+) from roots to shoots. The molecular structure and function of two classes of energised K(+) transporters are also reviewed. The first class is energised by Na(+) and shares structural similarities with K(+) transport mechanisms in bacteria and fungi. Structure-function studies suggest that it should be possible to increase the K(+) and Na(+) selectivity of these transporters, which will enhance the salt tolerance of higher plants. The second class of K(+) transporter is comprised of a large gene family and appears to have a dual affinity for K(+). A suite of molecular techniques, including gene cloning, oocyte expression, RNA localisation and gene inactivation, is now being used to fully characterise the biophysical and physiological function of plants K(+) transport mechanisms.     

Basolateral membrane K+ channels in renal epithelial cells

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K(+) channels play critical roles in normal physiology. Over 90 different genes for K(+) channels have been identified in the human genome. Epithelial K(+) channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K(+) channels is to recycle K(+) across the basolateral membrane for proper function of the Na(+)-K(+)-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K(+) channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a "K(+) channel gene family" approach in presenting the representative basolateral K(+) channels of the nephron. The basolateral K(+) channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families.     

上皮细胞K+通道的生理学意义与临床应用:跨上皮细胞转运的驱动力生成和K+循环

K^＋通道在上皮细胞内以极化的方式表达,形成一个庞大的膜蛋白家族。出于对主要依赖Na^＋-K^＋-ATPase而维持的细胞内跨膜K^＋梯度的考虑,K^＋通道在跨上皮细胞转运中的主要作用为：膜电位生成和K^＋循环。本文以肾近端小管和胃壁上皮细胞转运为例简要阐述了K^＋通道的作用。在这两个组织中,K^＋通道活性限速跨上皮细胞转运,调节细胞体积。近年来,药理学工具和转基因动物的实验证实了对K^＋通道的原先认知,并将研究深入到分子水平。K^＋通道的分子结构挑战高亲和力药物分子的设计,及其多组织同时表达的两个典型特征阻碍了高活性、组织特异性小分子治疗的进展。然而,抑制K^＋通道能阻断胃酸分泌等病理生理机制的深入研究,促进K^＋通道药物用于胃病治疗和作为肾脏转运抑制剂用于肾脏相关疾病治疗。     

A marriage of convenience: beta-subunits and voltage-dependent K+ channels

The movement of ions across cell membranes is essential for a wide variety of fundamental physiological processes, including secretion, muscle contraction, and neuronal excitation. This movement is possible because of the presence in the cell membrane of a class of integral membrane proteins dubbed ion channels. Ion channels, thanks to the presence of aqueous pores in their structure, catalyze the passage of ions across the otherwise ion-impermeable lipid bilayer. Ion conduction across ion channels is highly regulated, and in the case of voltage-dependent K(+) channels, the molecular foundations of the voltage-dependent conformational changes leading to the their open (conducting) configuration have provided most of the driving force for research in ion channel biophysics since the pioneering work of Hodgkin and Huxley (Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-544). The voltage-dependent K(+) channels are the prototypical voltage-gated channels and govern the resting membrane potential. They are responsible for returning the membrane potential to its resting state at the termination of each action potential in excitable membranes. The pore-forming subunits (alpha) of many voltage-dependent K(+) channels and modulatory beta-subunits exist in the membrane as one component of macromolecular complexes, able to integrate a myriad of cellular signals that regulate ion channel behavior. In this review, we have focused on the modulatory effects of beta-subunits on the voltage-dependent K(+) (Kv) channel and on the large conductance Ca(2+)- and voltage-dependent (BK(Ca)) channel.     

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

KCNE1 subunits require co-assembly with K+ channels for efficient trafficking and cell surface expression

KCNE peptides are a class of type I transmembrane beta subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K(+) channels. Accordingly, mutations that disrupt the assembly and trafficking of KCNE-K(+) channel complexes give rise to disease. The cellular mechanisms responsible for ensuring that KCNE peptides assemble with voltage-gated K(+) channels have yet to be elucidated. Using enzymatic deglycosylation, immunofluorescence, and quantitative cell surface labeling experiments, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with specific K(+) channel subunits; co-assembly mediates KCNE1 progression through the secretory pathway and results in cell surface expression. We also address an apparent discrepancy between our results and a previous study in human embryonic kidney cells, which showed wild type KCNE1 peptides can reach the plasma membrane without exogenously expressed K(+) channel subunits. By comparing KCNE1 trafficking in three cell lines, our data suggest that the errant KCNE1 trafficking observed in human embryonic kidney cells may be due, in part, to the presence of endogenous voltage-gated K(+) channels in these cells.     

Ion channels in opossum kidney cells.

This review discusses the activation of ion transport pathways during regulatory volume decrease in opossum kidney (OK) cells. OK cells regulate their volume when exposed to a hypotonic medium. The changes in cell volume are caused by activation of ion transport pathways and the accompanying osmotically driven water movement so that the increased cell volume returns toward physiological levels. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease. In OK cells separate K+ and Cl- conductances are activated. The Na+/H+ cotransport system seems not to be involved. The potassium pathway is mediated by a K+ channel with a slope conductance of about 12 pS. The occasionally observed widely distributed Ca2(+)- and voltage-dependent K+ channel of large unit conductance (120 pS) seems not to be involved. The volume regulatory decrease is accompanied by a cell depolarization from a resting potential of about -60 mV to about -20 mV followed by a repolarization. It will be discussed whether the depolarization is caused by the observed activation of stretch-sensitive ion channels of about 30 and 40 pS, respectively. The transient behavior of the cell volume parallels the time-dependent change of the total membrane current. For both recording techniques the volume regulatory decrease can be blocked by quinine. In addition an inward rectifying K+ channel of about 80 pS has been observed in high KCl solution.     

Plasma membrane ion channels in suicidal cell death

The machinery leading to apoptosis includes altered activity of ion channels. The channels contribute to apoptotic cell shrinkage and modify intracellular ion composition. Cl(-) channels allow the exit of Cl(-), osmolytes and HCO(3)(-) leading to cell shrinkage and cytosolic acidification. K(+) exit through K(+) channels contributes to cell shrinkage and decreases intracellular K(+) concentration, which in turn favours apoptotic cell death. K(+) channel activity further determines the cell membrane potential, a driving force for Ca(2+) entry through Ca(2+) channels. Ca(2+) may enter through unselective cation channels. An increase of cytosolic Ca(2+) may stimulate several enzymes executing apoptosis. Specific ion channel blockers may either promote or counteract suicidal cell death. The present brief review addresses the role of ion channels in the regulation of suicidal cell death with special emphasis on the role of channels in CD95 induced apoptosis of lymphocytes and suicidal death of erythrocytes or eryptosis.     

Phosphorylation of Ion channels

The introduction of highly specific reagents such as enzymes and inhibitors directly into living cells has proven to be a powerful tool in studying the modulation of cellular activity by protein phosphorylation. The use of exogenous kinases can be thought of as a pharmacological approach: this demonstrates that phosphorylation can produce modulation, but does not address the question of whether the cell actually uses this mechanism under normal physiological conditions. The complementary approach, the introduction of highly specific inhibitors such as R subunit or PKI, does ask whether endogenous kinase activity is necessary for a given physiological response. Together these two approaches have provided rather compelling evidence that cAMP-dependent and calcium/phospholipid-dependent protein phosphorylations can regulate membrane excitability. In several cases single-channel analysis has allowed the demonstration that an ion channel itself or something very close to the channel is the phosphorylation target, and it seems reasonable to assume that this will also be the case for many if not all of the other systems described above. Have any general principles emerged from the results to date? Certainly it seems clear that protein phosphorylation regulates not one but many classes of ion channels. As summarized in the Table, different channels can be modulated in different cells, some channels are activated while others are inhibited, and in some cells more than one channel is subject to modulation by phosphorylation. The list in the Table is probably not yet complete, and indeed it is not inconceivable that all ion channels can under appropriate conditions be regulated by phosphorylation. What aspect of channel function is altered by phosphorylation? The total membrane current, I, carried by a particular species of ion channel is given by Npi, where N is the number of active channels in the membrane, p is the probability that an individual channel will be open, and i is the single-channel current. In principle a change in I, the quantity measured in whole cell experiments, could be caused by a change in any one (or more) of the parameters, N, p or i (see Fig. 1). In the two cases in which single-channel measurements have allowed this question to be investigated, changes in N (Shuster et al., 1985) and p (Ewald et al., 1985) have been observed. Here again it seems unlikely that any one mechanism operates in all cases, and it would not be surprising to find that phosphorylation of some other channel results in a change in i.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.     

Periodic forcing of a K+ channel at various temperatures.

The random sequence of openings and closings of single ion channels and the channel conductances have been the object of intense study over the past two decades with a view toward illuminating the underlying kinetics of the channel protein molecules. Channels that are sensitive to voltage, such as many K(+)-selective channels, have been particularly useful, because the kinetic rates can be manipulated by changing the membrane voltage. Most such studies have been performed under stationary conditions and usually at a single temperature. Here we report the results of experiments with sinusoidal modulation of the membrane potential performed at several temperatures. Dwell time and cycle histograms, objects not normally associated with ion channel experiments, are herein reported. From the last, the transition probability densities for channel opening and closing events are obtained. A new and unusual phase anticipation is observed in the cycle histograms, and its temperature dependence is measured.     

Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae

The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K(+) channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K(+) (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K(+) channel function.     

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.     

Angiotensin II blocks ATP-sensitive K+ channels in porcine coronary artery smooth muscle cells.

ATP-sensitive K+ channels with small conductance (30 pS in symmetrical K(+)-rich solutions) in porcine coronary artery smooth muscle cells were highly active at physiological concentrations of Ca2+ (greater than 10(-4) M) even in the presence of physiological ATP levels, suggesting that these channels contribute to the generation of the resting membrane potential in vascular smooth muscle cells and their modulation is important in controlling vasomotor tone. Angiotensin II, applied from outside the membrane, blocked these channels in a concentration-dependent manner. This would be expected to cause depolarization and result in vasoconstriction.     

Molecular identification and physiological roles of parotid acinar cell maxi-K channels

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca(2+)-activated Cl(-) and K(+) channels. Parotid acinar cells express two types of Ca(2+)-activated K(+) channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the K(Ca)3.1 gene, and the K(Ca)1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the K(Ca)1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in K(Ca)1.1-null animals. We examined fluid secretion from animals with both K(+) channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K(+) channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.     

A BK (Slo1) channel journey from molecule to physiology

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca²⁺ and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca²⁺ sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.