Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae

1992 ◽  
Vol 338 (1283) ◽  
pp. 63-72 ◽  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Ion Channels ◽  
Open Channel ◽  
Single Channel ◽  
Membrane Channel ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Inside Out

Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na + or K + equally well, and Ca 2+ to a lesser extent. Its open probability (P„) is voltage-dependent, peaking at about — 80 mV (cytoplasm negative), and falling to near zero at + 8 0 mV. Elevated cytoplasmic Ca 2+ , alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K + over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca 2+ , this channel is activated by positive going membrane voltages: mean P o is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca 2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca 2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca 2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg 2+ of Na + , block the yeast plasma-membrane K + channel in a similar but less pronounced manner.

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.

scholarly journals Mechanism of Tacrine Block at Adult Human Muscle Nicotinic Acetylcholine Receptors

2002 ◽  
Vol 120 (3) ◽  
pp. 369-393 ◽  
Author(s):  
Richard J. Prince ◽  
Richard A. Pennington ◽  
Steven M. Sine
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Open Channel ◽  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Sequential Model ◽  
Hek 293 ◽  
Voltage Dependent

We used single-channel kinetic analysis to study the inhibitory effects of tacrine on human adult nicotinic receptors (nAChRs) transiently expressed in HEK 293 cells. Single channel recording from cell-attached patches revealed concentration- and voltage-dependent decreases in mean channel open probability produced by tacrine (IC50 4.6 μM at −70 mV, 1.6 μM at −150 mV). Two main effects of tacrine were apparent in the open- and closed-time distributions. First, the mean channel open time decreased with increasing tacrine concentration in a voltage-dependent manner, strongly suggesting that tacrine acts as an open-channel blocker. Second, tacrine produced a new class of closings whose duration increased with increasing tacrine concentration. Concentration dependence of closed-times is not predicted by sequential models of channel block, suggesting that tacrine blocks the nAChR by an unusual mechanism. To probe tacrine's mechanism of action we fitted a series of kinetic models to our data using maximum likelihood techniques. Models incorporating two tacrine binding sites in the open receptor channel gave dramatically improved fits to our data compared with the classic sequential model, which contains one site. Improved fits relative to the sequential model were also obtained with schemes incorporating a binding site in the closed channel, but only if it is assumed that the channel cannot gate with tacrine bound. Overall, the best description of our data was obtained with a model that combined two binding sites in the open channel with a single site in the closed state of the receptor.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart

1990 ◽  
Vol 259 (5) ◽  
pp. H1609-H1612 ◽  
Author(s):  
A. I. Undrovinas ◽  
N. Burnashev ◽  
D. Eroshenko ◽  
I. Fleidervish ◽  
C. F. Starmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Pipette Solution ◽  
Channel Current ◽  
Closed Intervals ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Inside Out ◽  
Ischemic Arrhythmias

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C733-C738 ◽  
Author(s):  
L. Vaca ◽  
D. L. Kunze
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Single Channel ◽  
Vascular Endothelial Cells ◽  
Channel Activity ◽  
Open Probability ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Selective Channel ◽  
Inside Out

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

Basolateral outward rectifier chloride channel in isolated crypts of mouse colon

2000 ◽  
Vol 279 (2) ◽  
pp. G277-G287 ◽  
Author(s):  
Olivier Mignen ◽  
Stéphane Egee ◽  
Martine Liberge ◽  
Brian J. Harvey
Keyword(s):  
Protein Kinase ◽  
Chloride Channels ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Open Probability ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Inside Out

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between −100 and −40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I− > SCN− > Br−> Cl− > NO3 − > F−≫ SO4 2− ≈ gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca2+ concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5′- O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5′- O-(2-thiodiphosphate) reactivated the channel.

scholarly journals Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies.

1992 ◽  
Vol 172 (1) ◽  
pp. 271-287
Author(s):  
A Bertl ◽  
C L Slayman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Single Channel ◽  
K Channel ◽  
Substrate Uptake ◽  
Alkaline Ph ◽  
Open Probability ◽  
Extracellular Medium ◽  
Yeast Saccharomyces Cerevisiae ◽  
Charge Balancing

Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.

scholarly journals Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

2002 ◽  
Vol 120 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Ion Channels ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Acid Sensing Ion Channels ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetics Of ◽  
Channel Properties

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small.

scholarly journals Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

1998 ◽  
Vol 111 (2) ◽  
pp. 363-379 ◽  
Author(s):  
Izumi Sugihara
Keyword(s):  
Dissociation Constant ◽  
Time Constant ◽  
Hair Cells ◽  
Single Channel ◽  
Hill Coefficient ◽  
K Channels ◽  
Voltage Dependent ◽  
Time Courses ◽  
The Hill ◽  
Inside Out

Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (>100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).

scholarly journals The Na+/Ca2+ Exchanger Inhibitor, KB-R7943, Blocks a Nonselective Cation Channel Implicated in Chemosensory Transduction

2009 ◽  
Vol 101 (3) ◽  
pp. 1151-1159 ◽  
Author(s):  
A. Pezier ◽  
Y. V. Bobkov ◽  
B. W. Ache
Keyword(s):  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Trpc Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Olfactory Transduction ◽  
Voltage Dependent ◽  
Chemosensory Transduction ◽  
Transient Receptor

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na+/Ca2+ exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na+/Ca2+ exchanger.

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