Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2 3 cyclic nucleotide 3 phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.     

Three Types of Single Voltage-Dependent Potassium Channels in the Sarcolemma of Frog Skeletal Muscle

Patch-clamp experiments in the sarcolemma of frog skeletal muscle evidenced the presence of three types of voltage-dependent single-channel K⁺ currents. According to their unitary conductance at a membrane voltage of +40 mV, we classified them as 16-, 13-, and 7-pS K⁺ channels. The 16-pS K⁺ channels are active close to a membrane voltage of −80 mV and they do not become inactivated during voltage pulses of 100 ms. Within 10 min after beginning the recording, these channels developed rundown with an exponential time course. The 13-pS K⁺ channels are active near −60 mV; upon a 100-ms depolarization, they exhibited inactivation with an approximate exponential time course. The 7-pS K⁺ channels were recorded at voltages positive to 0 mV. In patches containing all three types of K⁺ channels, the ensemble average currents resemble the kinetic properties of the macroscopic delayed rectifier K⁺ currents recorded in skeletal muscle and other tissues. In conclusion, the biophysical properties of unitary K⁺ currents suggest that these single-channel K⁺ currents may underlie the macroscopic delayed K⁺ currents in frog skeletal muscle fibers. In addition, since the 16- and 13-pS channels were more frequently recorded, both are the main contributors to the delayed K⁺ currents.     

Genistein Inhibits the Activity of Kv1.3 Potassium Channels in Human T Lymphocytes

In the present study, the whole-cell patch-clamp technique was applied to follow the inhibitory effect of genistein — a tyrosine kinase inhibitor and a natural anticancer agent—on the activity of voltage-gated potassium channels Kv1.3 expressed in human T lymphocytes (TL). Obtained data provide evidence that genistein application in the concentration range of 1–80 μM reversibly decreased the whole-cell potassium currents in TL in a concentration-dependent manner to about 0.23 of the control value. The half-blocking concentration range of genistein was from 10 to 40 μM. The current inhibition was correlated in time with a significant decrease of the current activation rate. The steady-state activation of the currents was unchanged upon application of genistein, as was the inactivation rate. The inhibitory effect of genistein on the current amplitude and activation kinetics was voltage-independent. The current inhibition was not changed significantly in the presence of 1 mM of sodium orthovanadate, a tyrosine phosphatase inhibitor. Application of daidzein, an inactive genistein analogue, did not affect significantly either the current amplitudes or the activation kinetics. Possible mechanisms of the observed phenomena and their significance for genistein-induced inhibition of cancer cell proliferation are discussed.     

Inhibition of the Activity of Human Lymphocyte Kv1.3 Potassium Channels by Resveratrol

The whole-cell patch-clamp technique was applied to study the modulatory effect of resveratrol on voltage-gated potassium channel Kv1.3 expressed in human lymphocytes. Results demonstrate that application of resveratrol in the concentration range 1–200 μM inhibited the channel activity in a concentration-dependent manner to about 18% of the control value. The half-blocking concentration of resveratrol was 40.9 μM, whereas the Hill coefficient was 1.05. The inhibition was time-dependent and slowly reversible. The inhibitory effect of resveratrol was correlated in time with a significant slowing of the current activation, whereas the inactivation rate remained unaffected upon application of resveratrol. The inhibition of Kv1.3 channels was voltage-independent. The steady-state activation of the currents remained unchanged upon resveratrol application. The magnitude of the inhibitory effect of resveratrol was not altered when resveratrol was coapplied with genistein. The possible mechanism of the inhibitory effect and its significance for biological activity of resveratrol are discussed.     

Subunit Stoichiometry of Human Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl⁻ channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl⁻ channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

N-terminal PDZ-binding domain in Kv1 potassium channels

We have investigated the interactions of prototypical PDZ domains with both the C- and N-termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two-hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C- and N-termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co-immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C-terminal PDZ-binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co-localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ-binding motif. Deletion analysis localized the N-terminal PDZ-binding site in Kv1.5 to the T1 region of the channel. Co-expression of PSD95 with Kv1.5 N- and C-terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95.     

The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate possible expression of Kv1.3 channels in other types of tissue, such as epithelia, binding experiments, immunoprecipitation studies and immunohistochemical studies were performed. The double-mutated, radiolabeled peptidyl ligand, ¹²⁵I-HgTX₁-A19Y/Y37F, which selectively binds Kv1.1, Kv1.2, Kv1.3 and Kv1.6 channels, was used to perform binding studies in epithelia isolated from rabbit kidney and colon. The equilibrium dissociation constant for this ligand was found to be in the sub-picomolar range and the maximal receptor concentration (in fmol/mg protein) 1.68 for colon and 0.61-0.75 for kidney epithelium. To determine the subtype of Kv1 channels, immunoprecipitation studies with ¹²⁵I-HgTX₁-A19Y/Y37F labeled epithelial membranes were performed with specific antibodies against Kv1.1, Kv1.2, Kv1.3, Kv1.4 or Kv1.6 subunits. These studies demonstrated that Kv1.3 subunits constituted more than 50% of the entire Kv1 subunit population. The precise localization of Kv1.3 subunits in epithelia was determined by immunohistochemical studies.     

Reduced Kv1.3 Potassium Channel Expression in Human Prostate Cancer

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = −0.25, P = 0.003) as well as tumor stage (r = −0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.     

The Presence of Phospholipase D In Rat Central Nervous System Axolemma

An axolemma-enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma-enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.     

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.     

Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels

The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.     

Homology Models of the Tetramerization Domain of Six Eukaryotic Voltage-gated Potassium Channels Kv1.1-Kv1.6

Abstract

The homology models of the tetramerization (T1) domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were constructed based on the crystal structure of the Shaker T1 domain. The results of amino acid sequence alignment indicate that the T1 domains of these K⁺ channels are highly conserved, with the similarities varying from 77% between Shaker and Kv1.6 to 93% between Kv1.2 and Kv1.3. The homology models reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K⁺ channel. These models also show that each T1 monomer consists of three distinct layers, with N-terminal layer 1 and C- terminal layer 3 facing the cytoplasm and the membrane, respectively. Layer 2 exhibits the highest structural conservation because it is located around the central hydrophobic core. For each Kv channel, four identical subunits assemble into the homotetramer architecture around a four-fold axis through the hydrogen bonds and salt bridges formed by 15 highly conserved polar residues. The narrowest opening of the pore is formed by the four conserved residues corresponding to R115 of the Shaker T1 domain. The homology models of these Kv T1 domains provide particularly attractive targets for further structure-based studies.     

Kinetic properties of Kv4.3 and their modulation by KChIP2b

KChIPs are a family of Kv4 K(+) channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K(+) channels.     

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.     

Voltage-activated outward currents in adult and nymphal locust photoreceptors

The membrane properties of dark-adapted green sensitive photoreceptors of adult and nymphal desert locust (Schistocerca gregaria) were investigated in situ with single electrode (discontinuous) current and voltage clamp techniques. The photoreceptors had a resting potential of about -70 mV. The membrane rectified strongly in response to depolarizing current pulses at and above the resting potential. Two outward currents could be distinguished on the basis of different kinetics. The first, a transient current, was inactivating between-40 mV and -100 mV, and activated above -90 mV. The second, a sustained current had a similar activation range. The inactivating current could be blocked by application of 50 mM TEA into the retinal extracellular space. Both currents were expressed in nymphal photoreceptors already in the 1st instar larva and have similar properties as in the adults, although the behaviour of adults and nymphs is different. The conductances underlying these currents could be shown to modify the frequency response of the photoreceptor membrane.The work was started in the Department of Zoology, University of Cambridge, Cambridge, UK.     

Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K⁺ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.     

Src regulates membrane trafficking of the Kv3.1b channel

The Kv3.1 channel plays a crucial role in regulating the high-frequency firing properties of neurons. Here, we determined whether Src regulates the subcellular distributions of the Kv3.1b channel. Co-expression of active Src induced a dramatic redistribution of Kv3.1b to the endoplasmic reticulum. Furthermore, co-expression of the Kv3.1b channel with active Src induced a remarkable decrease in the pool of Kv3.1b at the cell surface. Moreover, the co-expression of active Src results in a significant decrease in the peak current densities of the Kv3.1b channel, and a substantial alteration in the voltage dependence of its steady-state inactivation. Taken together, these results indicate that Src kinase may play an important role in regulating membrane trafficking of Kv3.1b channels.     

槲皮素对谷氨酸诱导的PC12细胞损伤的保护作用及对海马CA1锥体神经元钾通道的影响(英文)

槲皮素广泛存在于许多药用植物中,属黄酮类化合物,在临床上常用于心血管疾病的治疗。采用四甲基偶氮唑盐比色法(MTT法)及DAPI染色,研究槲皮素(0.5、1、5、10μmol/L)对谷氨酸(10mmol/L)诱导的PC12细胞损伤作用的影响;并进一步研究槲皮素(0.3、3、30μmol/L)对急性分离的海马CA1锥体神经元离子通道的作用。MTT实验结果显示,槲皮素可提高谷氨酸处理组PC12细胞的存活率,并呈现为浓度和时间依赖性(P0.05);而槲皮素(5μmol/L)与谷氨酸(10mmol/L)共孵育PC12细胞后,DAPI染色结果表明槲皮素可减弱谷氨酸对PC12细胞的损伤。对电生理结果显示,槲皮素对瞬时外向钾电流(IA)和延迟整流钾电流(IK)有显著的抑制作用(P0.05),表现为浓度依赖性。以上结果提示,槲皮素可能通过抑制海马锥体神经元的外向钾电流进而对谷氨酸诱导的神经损伤起保护作用,这也说明了槲皮素对缺血样损伤的神经具有保护作用。