scholarly journals Chlorella virus ATCV-1 encodes a functional potassium channel of 82 amino acids

2009 ◽  
Vol 420 (2) ◽  
pp. 295-305 ◽  
Author(s):  
Sabrina Gazzarrini ◽  
Ming Kang ◽  
Alessandra Abenavoli ◽  
Giulia Romani ◽  
Claudio Olivari ◽  
...  
Keyword(s):  
Amino Acids ◽  
Potassium Channel ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
K Channel ◽  
Channel Blockers ◽  
Selective Filter ◽  
Chlorella Virus ◽  
Charged Amino Acids ◽  
Voltage Dependent

Chlorella virus PBCV-1 (Paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In this paper, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisiae mutants that lack endogenous K+ uptake systems. Compared with the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, barium and caesium. Single channel analyses reveal a stochastic gating behaviour and a voltage-dependent conductance that resembles the macroscopic I/V relationship. One difference between PBCV-1 and ATCV-1 Kcv is that the latter is more permeable to K+ than Rb+. This difference is partially explained by the presence of a tyrosine residue in the selective filter of ATCV-1 Kcv, whereas PBCV-1 Kcv has a phenylalanine. Hence, ATCV-1 Kcv is the smallest protein to form a K+ channel and it will serve as a model for studying structure–function correlations inside the potassium channel pore.

scholarly journals Further studies of ion channels in the electroreceptor of the skate through deep sequencing, cloning and cross species comparisons

2018 ◽  
Author(s):  
William T. Clusin ◽  
Ting-Hsuan Wu ◽  
Ling-Fang Shi ◽  
Peter N. Kao
Keyword(s):  
Amino Acids ◽  
Ion Channels ◽  
Potassium Channel ◽  
Calcium Channel ◽  
Beta Subunit ◽  
K Channel ◽  
Rna Seq ◽  
Α Subunit ◽  
Accessory Subunits ◽  
Voltage Dependent

AbstractOur comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel, whose α subunit is Kcnma1, a voltage-dependent calcium channel, Cacna1d, and a relatively uncharacterized K channel which interacts with the calcium channel to produce fast (20 Hz) oscillations. Large conductance calcium-activated K channels (BK) are comprised of four α subunits, encoded by Kcnma1 and modulatory β subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 derived from using purified mRNA of homogenized isolated electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the the Bellono et al RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent potassium channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels and seems likely to explain the 20 Hz oscillations believed to occur in vivo. The second was more similar to Kv1.5 than to Kv1.1 but was somewhat atypical. We also found a beta subunit sequence (Kcnab2) which appears not to cause fast inactivation due to specific structural features. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of the calcium activated potassium channel, Kcnma1, and found a computer generated assembly that contained a complete sequence of its beta subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 (beta1) is absent. The evolutionary origin of the newly described channels and subunits was studied by aligning skate sequences with human sequences and those found in related fish: the whale shark (R. typus) an elasmobranch, and ghost shark (C.milii). There is also homology with the lamprey, which has electroreceptors. An evolutionary tree is presented. Further research should include focusing on the subcellular locations of these channels in the receptor cells, their gating behavior, and the effects of accessory subunits on gating.

Charged amino acids near the H5-region and channel functions in voltage-dependent potassium channel, Kv1.5

1994 ◽  
Vol 19 ◽  
pp. S11
Author(s):  
Tetsushi Furukawa ◽  
Yoshifumi Katayama ◽  
Tei-Ichi Yamane ◽  
Masayasu Hiraoka
Keyword(s):  
Amino Acids ◽  
Potassium Channel ◽  
Charged Amino Acids ◽  
Voltage Dependent

scholarly journals Functional Roles of Charged Amino Acid Residues on the Wall of the Cytoplasmic Pore of Kir2.1

2006 ◽  
Vol 127 (4) ◽  
pp. 401-419 ◽  
Author(s):  
Yuichiro Fujiwara ◽  
Yoshihiro Kubo
Keyword(s):  
Amino Acids ◽  
Negative Charge ◽  
Single Channel ◽  
Outward Current ◽  
Channel Noise ◽  
Inward Rectification ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Charged Amino Acids ◽  
Charged Residues

It is known that rectification of currents through the inward rectifier K+ channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg2+ and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K+ permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg2+ or spermine and the voltage dependence of the blockade. It also reduced K+ permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg2+, spermine, and K+ so that the transmembrane pore is sufficiently filled with K+ ions, which enables strong voltage-dependent blockade with adequate outward K+ conductance.

Ionic channels in corneal endothelium

1996 ◽  
Vol 270 (4) ◽  
pp. C975-C989 ◽  
Author(s):  
J. L. Rae ◽  
M. A. Watsky
Keyword(s):  
Patch Clamp ◽  
Corneal Endothelium ◽  
Single Channel ◽  
Cell Voltage ◽  
Anion Channel ◽  
Ionic Channels ◽  
K Channel ◽  
Na Channel ◽  
Channel Blockers ◽  
K Currents

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

scholarly journals Action Potentials in Rhodnius Oocytes: Repolarization is Sensitive to Potassium Channel Blockers

1986 ◽  
Vol 126 (1) ◽  
pp. 119-132
Author(s):  
M. J. O'DONNELL
Keyword(s):  
Potassium Channel ◽  
Action Potentials ◽  
Potassium Conductance ◽  
Channel Blockers ◽  
Calcium Dependent ◽  
Outward Rectification ◽  
Current Voltage ◽  
Potassium Channel Blockers ◽  
Potassium Conductances ◽  
Voltage Dependent

Depolarization of Rhodnius oocytes evokes action potentials (APs) whose rising phase is calcium-dependent. The ionic basis for the repolarizing (i.e. falling) phase of the AP was examined. Addition of potassium channel blockers (tetraethylammonium, tetrabutylammonium, 4-aminopyridine, atropine) to the bathing saline increased the duration and overshoot of APs. Intracellular injection of tetraethyl ammonium had similar effects. These results suggest that a voltage-dependent potassium conductance normally contributes to repolarization. Repolarization does not require a chloride influx, because substitution of impermeant anions for chloride did not increase AP duration. AP duration and overshoot actually decreased progressively when chloride levels were reduced. Current/voltage curves show inward and outward rectification, properties often associated with potassium conductances. Outward rectification was largely blocked by external tetraethylammonium. Possible functions of the rectifying properties of the oocyte membrane are discussed.

scholarly journals K+ channel blockade in the NTS alters efficacy of two cardiorespiratory reflexes in vivo

1998 ◽  
Vol 274 (3) ◽  
pp. R677-R685 ◽  
Author(s):  
James W. Butcher ◽  
Julian F. R. Paton
Keyword(s):  
Potassium Channel ◽  
Neuronal Excitability ◽  
Baroreceptor Reflex ◽  
Right Atrial ◽  
K Channel ◽  
Reflex Bradycardia ◽  
Potassium Conductances ◽  
Voltage Dependent

We investigated the role of potassium conductances in the nucleus of the solitary tract (NTS) in determining the efficacy of the baroreceptor and cardiopulmonary reflexes in anesthetized rats. The baroreceptor reflex was elicited with an intravenous injection of phenylephrine to evoke a reflex bradycardia, and the cardiopulmonary reflex was evoked with a right atrial injection of phenylbiguanide. Microinjection of two Ca-dependent potassium channel antagonists (apamin and charybdotoxin) into the NTS potentiated the baroreceptor reflex bradycardia. This may reflect the increased neuronal excitability observed previously in vitro with these blockers. In contrast, the Ca-dependent potassium channel antagonists attenuated the cardiopulmonary reflex, whereas voltage-dependent potassium channel antagonists (4-aminopyridine and dendrotoxin) attenuated both the baro- and cardiopulmonary reflexes when microinjected into the NTS. The possibility that the reflex attenuation observed indicates a predominant distribution of certain potassium channels on γ-aminobutyric acid interneurons is discussed.

scholarly journals Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1

2002 ◽  
Vol 120 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Lai-Hua Xie ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Single Channel ◽  
Quantitative Model ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Surface Charges ◽  
Open Probability ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inside Out

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Complexity of the outward K+ current of the rat megakaryocyte

1997 ◽  
Vol 272 (5) ◽  
pp. C1525-C1531 ◽  
Author(s):  
E. Romero ◽  
R. Sullivan
Keyword(s):  
K Channel ◽  
Delayed Rectifier ◽  
Channel Blockers ◽  
Excitable Cells ◽  
Electrophysiological Properties ◽  
Relative Contribution ◽  
Delayed Rectifier Current ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
K Current

Megakaryocytes isolated from rat bone marrow express a voltage-dependent, outward K+ current with complex kinetics of activation and inactivation. We found that this current could be separated into at least two components based on differential responses to K+ channel blockers. One component, which exhibited features of the "transient" or "A-type" K+ current of excitable cells, was more strongly blocked by 4-aminopyridine (4-AP) than by tetrabutylammonium (TBA). This current, which we designated as "4-AP-sensitive" current, activated rapidly at potentials more positive than -40 mV and subsequently underwent rapid voltage-dependent inactivation. A separate current that activated slowly was blocked much more effectively by TBA than by 4-AP. This "TBA-sensitive" component, which resembled a typical delayed rectifier current, was much more resistant to voltage-dependent inactivation. The relative contribution of each of these components varied from cell to cell. The effect of charybdotoxin was similar to that of 4-AP. Our data indicate that the voltage-dependent K+ current of resting megakaryocytes is more complex than heretofore believed and support the emerging concept that megakaryocytes possess intricate electrophysiological properties.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Characterization of a novel 132-bp exon of the human maxi-K channel

2001 ◽  
Vol 281 (1) ◽  
pp. C361-C367 ◽  
Author(s):  
Victoria P. Korovkina ◽  
Daniel J. Fergus ◽  
Amanda J. Holdiman ◽  
Sarah K. England
Keyword(s):  
Alternative Splicing ◽  
Single Channel ◽  
K Channel ◽  
Distribution Analysis ◽  
Protein Distribution ◽  
Intracellular Loop ◽  
Cell Excitability ◽  
Muscle Tissues ◽  
Voltage Dependent ◽  
Intracellular Ca

The large-conductance Ca2+-activated voltage-dependent K+ channel (maxi-K channel) induces a significant repolarizing current that buffers cell excitability. This channel can derive its diversity by alternative splicing of its transcript-producing isoforms that differ in their sensitivity to voltage and intracellular Ca2+. We have identified a novel 132-bp exon of the maxi-K channel from human myometrial cells that encodes 44 amino acids within the first intracellular loop of the channel protein. Distribution analysis reveals that this exon is expressed predominantly in human smooth muscle tissues with the highest abundance in the uterus and aorta and resembles the previously reported distribution of the total maxi-K channel transcript. Single-channel K+ current measurements in fibroblasts transfected with the maxi-K channel containing this novel 132-bp exon demonstrate that the presence of this insert attenuates the sensitivity to voltage and intracellular Ca2+. Alternative splicing to introduce this 132-bp exon into the maxi-K channel may elicit another mode to modulate cell excitability.

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