Differential Asparagine-Linked Glycosylation of Voltage-Gated K+ Channels in Mammalian Brain and in Transfected Cells

Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked glycosylation of voltage-gated K⁺ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels, reflecting the different efficiencies of transport of K⁺ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich N-linked oligosaccharide chains differs among highly related K⁺ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential glycosylation contributing to diversity of K⁺ channel function in mammalian brain. Received: 17 December 1998/Accepted: 20 January 1999     

K+ and Na+ Conduction in Selective and Nonselective Ion Channels Via Molecular Dynamics Simulations

Generations of scientists have been captivated by ion channels and how they control the workings of the cell by admitting ions from one side of the cell membrane to the other. Elucidating the molecular determinants of ion conduction and selectivity are two of the most fundamental issues in the field of biophysics. Combined with ongoing progress in structural studies, modeling and simulation have been an integral part of the development of the field. As of this writing, the relentless growth in computational power, the development of new algorithms to tackle the so-called rare events, improved force-field parameters, and the concomitant increasing availability of membrane protein structures, allow simulations to contribute even further, providing more-complete models of ion conduction and selectivity in ion channels. In this report, we give an overview of the recent progress made by simulation studies on the understanding of ion permeation in selective and nonselective ion channels.     

不同抗盐性小麦叶鞘的Na+/K+选择性

以200mmol／LNaCl处理耐盐小麦（Triticum aestivum L．）品种DK961和盐敏感小麦品种LM153d后,DK961叶鞘中Na^＋含量和叶片中K^＋含量均显著高于LMl5,这造成DK961叶片中Na^＋／K^＋比显著低于LM15。即DK961的叶鞘以其较LM15更强的限制Na^＋、推动K^＋向叶片运输的能力来保持叶片相对较低的Na^＋／K^＋比,即叶鞘在小麦抗盐性中可能起重要作用。     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios

The capacity of plants to maintain a high cytosolic K⁺/Na⁺ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K⁺/Na⁺ratio. For K⁺uptake,K⁺efflux and K⁺translocation to the shoot, genes have been isolatedthat encode K⁺uptake and K⁺release ion channels and K⁺carriersthat are coupled to either a H⁺or Na⁺gradient. Although thepicture is less clear for the movement of Na⁺, one pathway,in the form of non-selective ion channels, is likely to playa role in Na⁺uptake, whereas Na⁺efflux and compartmentationare likely to be mediated by H⁺-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K⁺and/or Na⁺fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K⁺nutritionand Na⁺toxicity. Copyright 1999 Annals of Botany Company Salinty, K⁺/N⁺ratio, transporter, membrane.     

Alternative Mechanisms for Fast Na+/Ca2+ Signaling in Eukaryotes via a Novel Class of Single-Domain Voltage-Gated Channels

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Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.     

Role of Voltage-Gated K+ (KV) Channels in Vascular Function

Voltage-dependent K⁺ (K_V) channels represent the most diverse group of K⁺ channels ubiquitously expressed in vascular smooth muscles. The K_V channels, together with other types of K⁺ conductances, such as Ca²⁺-activated (BK_Ca), ATP-sensitive (K_ATP), and inward rectifier, play an important role in the control of the cell membrane potential and regulation of the vascular contractility. Comparison of the expression of different K_V channel isoforms obtained from RT-PCR studies showed that virtually all K_V genes could be detected in vascular smooth muscle cells (VSMC). Based on the analysis of both mRNA and protein expressions, it is likely that K_V1.1, K_V1.2, K_V1.3, K_V1.5, K_V1.6, K_V2.1, and K_V3.1b channel isoforms are mainly responsible for the delayed rectifier current characterized electrophysiologically in most VSMC types studied to date. It has been recently demonstrated by our research group and by others that functional expression of multiple K_V channel α-subunits is not homogeneous and varies in different vascular beds of small and large arteries. Growing evidence suggests that in some small arteries, e.g., cerebral arteries and arterioles, the K_V channels are activated at more negative membrane voltages than BK_Ca, thus making a greater contribution to the control of vascular tone. Our data also suggest that in some blood vessels, such as the rat aorta and mouse small mesenteric arteries, the K_V channel current (identified mainly as passed through K_V2.1 channels), but not BK_Ca, is the predominant conductance activated even under conditions where intracellular Ca₂₊ concentration is increased up to 200 nM. In addition, our data indicate that the K_V2.1 channel current could also contribute to the regulation of the induced rhythmic activity in the rat aorta in vitro acting as a negative feedback mechanism for membrane depolarization. We and other experimenters also demonstrated that functional expression of K_V channels is a dynamic process, which is altered under normal physiological conditions (e.g., during the development of the vessels), and in various pathological states (e.g., pulmonary hypertension developing during chronic hypoxia). Recent findings also suggest that activation of K_V channels can also play a role in vascular apoptosis (causing loss of intracellular K⁺ and subsequent cell shrinking, one of the essential prerequisites of cellular apoptosis). To summarize, the K^V channels are essential for normal vascular function, and their expression and properties are altered under abnormal conditions. Therefore, understanding of the molecular identity of native K_V channels and their functional significance and elucidation of the mechanisms, which govern and control the expression of the K_V channels in the vasculature, represent an important and challenging task and could also lead to the development of useful therapeutic strategies for the treatment of cardiovascular diseases.     

Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes

A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na⁺/K⁺ pump to intracellular Na⁺, whereas a large increase in cholesterol levels decreases the sensitivity to Na⁺. To examine the mechanisms, we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped with the use of patch pipettes that contained osmotically balanced solutions with Na⁺ in a concentration of 10 mM and K⁺ in concentrations ([K⁺]_pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na⁺/K⁺ current (I_p) when pipette solutions were K⁺ free. A modest increase in serum cholesterol caused a [K⁺]_pip-dependent increase in I_p, whereas a large increase caused a [K⁺]_pip-dependent decrease in I_p. Modeling suggested that pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant K_K describing the backward reaction E₁ + 2K⁺ E₂(K⁺)₂, whereas pump inhibition with a large increase in serum cholesterol can be explained by an increase in K_K. Because hypercholesterolemia upregulates angiotensin II receptors and because angiotensin II regulates the Na⁺/K⁺ pump in cardiac myocytes in a [K⁺]_pip-dependent manner, we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Because the -isoform of protein kinase C (PKC) mediates effects of angiotensin II on the pump, we included specific PKC-blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition. partial reactions; protein kinase C; angiotensin converting enzyme inhibitors; arteriosclerosis; insulin resistance     

K and Na Conduction in Selective and Nonselective Ion Channels Via Molecular Dynamics Simulations

Generations of scientists have been captivated by ion channels and how they control the workings of the cell by admitting ions from one side of the cell membrane to the other. Elucidating the molecular determinants of ion conduction and selectivity are two of the most fundamental issues in the field of biophysics. Combined with ongoing progress in structural studies, modeling and simulation have been an integral part of the development of the field. As of this writing, the relentless growth in computational power, the development of new algorithms to tackle the so-called rare events, improved force-field parameters, and the concomitant increasing availability of membrane protein structures, allow simulations to contribute even further, providing more-complete models of ion conduction and selectivity in ion channels. In this report, we give an overview of the recent progress made by simulation studies on the understanding of ion permeation in selective and nonselective ion channels.     

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.     

P-Loop Residues Critical for Selectivity in K+ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

HCN channels are thought to be structurally similar to K_v channels, but show much lower selectivity for K⁺. The ∼3.3 Å selectivity filter of K⁺ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L₄₇₈C₄₇₉IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K⁺ channels, we also studied C479T channels. Based on permeability ratios (P_X/P_K), C479T HCN4 channels (K⁺(1)>Rb⁺(0.85)>Cs⁺(0.59)>Li⁺(0.50)≥Na⁺(0.49)) were less selective than WT rabbit HCN4 (K⁺(1)>Rb⁺(0.48)>Cs⁺(0.31)≥Na⁺(0.29)>Li⁺(0.03)), indicating that the TIGYG sequence is insufficient to confer K⁺ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K⁺ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K⁺ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K⁺ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K⁺ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K⁺ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K⁺ channels originate from differences outside the P-loop region.     

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K⁺) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K⁺ channel type segregates into at least two main functional categories—hyperpolarization-activated, inward-rectifying (K_in) and depolarization-activated, outward-rectifying (K_out) channels. Voltage-gated K⁺ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in K_in and K_out channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K⁺ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between K_in and K_out channels. Among them is a loop between TMD S5 and the pore that is very short in animal K_out, longer in plant and animal K_in and the longest in plant K_out channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

K+-Na+ Exchange and Selectivity in Barley Root Cells: Effect of Na+ on the Na+ Fluxes

Using the compartmental analysis the unidirectional Na⁺ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa⁺ contents Q_c and Q_v, and the trans-root Na⁺ transport R'have been studied as a function of the external Na⁺ concentration.Using the re-elution technique the effect of low K⁺ concentrationson the plasmalemma efflux _co of Na⁺ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K⁺-Na⁺exchange system. The apparent Km corresponded to a cytoplasmicNa⁺ concentration of 28 mM at 0·2 mM K⁺ and about 0·2mM Na⁺ in the external solution. 0·2 mM K⁺ stimulatedthe plasma-lemma efflux of Na⁺ and inhibited Na⁺ transport selectivelyeven in the presence of 10 mM Na⁺ in the external medium showingthe high efficiency of the K⁺-Na⁺ exchange system. However,_co, K⁺-dep was inhibited at 10 mM Na¹ compared to lower Na¹concentrations suggesting some competition of Na¹ with K¹ atthe external site of the exchange system. The effect of theNa+ concentration on Na¹ influx _oc is discussed with respectto kinetic models of uuptake.     

The pathway for spontaneous occlusion of Rb+ in the Na+/K+-ATPase

Occlusion of K (+) in the Na (+)/K (+)-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na (+) and Mg (2+), after the K (+)-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K (+) to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb (+) in an unsided, purified preparation of Na (+)/K (+)-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K (+) and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg (2+) plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg (2+) plus Pi, exchange of K (+) would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb (+) ions was labeled as (86)Rb (+). We found that, when the enzyme is in the E2 conformation, the first Rb (+) that entered the enzyme in media without Mg (2+) and Pi was the last to leave after addition of Mg (2+) plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb (+) between E2(Rb 2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K (+) during the ATPase activity.