Phosphate and slow vacuolar channels in Beta vulgaris

1998 ◽  
Vol 25 (6) ◽  
pp. 709 ◽  
Author(s):  
James Dunlop ◽  
Thai Phung
Keyword(s):  
Ion Channels ◽  
Beta Vulgaris ◽  
Single Channel ◽  
Reversal Potential ◽  
Individual Species ◽  
Phosphate Ions ◽  
Current Voltage ◽  
Total Phosphate Concentration ◽  
Marked Dependence ◽  
Rate Of Activation

Movement of phosphate through slow vacuolar (SV) ion channels and the effects of phosphate on SV currents were investigated using vacuoles from Beta vulgaris L. When the vacuoles contained 50 mM phosphate, the addition of phosphate to the bath shifted the apparent reversal potential for whole vacuole currents to more positive values, suggesting an outward rectifying current due to movement of phosphate ions out of the vacuole. However absolute values for reversal potentials obtained from the current-voltage curves and tail currents for whole vacuoles suggested that the membranes were relatively impermeable to phosphate. Single-channel data showed that the vacuole preparations contained more than one species of ion channel and therefore the whole-vacuole data will not give definitive information about individual species of ion channels. One of the channels had a single channel reversal potential that indicated a permeability of H2PO4- ions relative to Cl- of 7. The probability of this outwardly rectifying channel being open had a marked dependence on voltage and, in these experiments, it was effectively closed for potentials negative of +20 mV. The single-channel conductance was 19.4 ± 3.1 pS with 50 mM KH2PO4 in the vacuole and 10 mM total phosphate concentration in the bath. A channel with these characteristics has not been reported previously. In addition to the data identifying a phosphate channel, it was found that the presence of phosphate in the bath solution slowed the rate of activation of the SV currents. This effect was partially reversed when phosphate was removed from the bath.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

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.

Ion channels in normal human and cystic fibrosis sweat gland cells

1989 ◽  
Vol 257 (1) ◽  
pp. C129-C140 ◽  
Author(s):  
M. E. Krouse ◽  
G. Hagiwara ◽  
J. Chen ◽  
N. J. Lewiston ◽  
J. J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Ion Channels ◽  
Sweat Gland ◽  
Single Channel ◽  
Cell Types ◽  
Cation Channels ◽  
Secretory Cells ◽  
Anion Channels ◽  
Current Voltage ◽  
Normal Human

Single-channel patch-clamp techniques were used to study the population of apical membrane ion channels in cultured sweat gland secretory cells from normal and cystic fibrosis subjects. Four types of anion channels and two types of cation channels were found. At physiological voltages, anion channels had chord conductances of 10, 18, 24, and greater than 200 pS. All had linear current-voltage relations except the 24 pS channel, which showed outward rectification. Cation channels had chord conductances of 5 and 18 pS, were linear, and were nonselective for a variety of cations. Channel types and proportions were equivalent in control, cystic fibrosis, and cystic fibrosis heterozygote cells. Beyond showing that the distribution of channel types remains unchanged in cystic fibrosis cells, the data provide a basis for comparison with cells cultured under different conditions, with other cell types, and with native tissues.

Capsaicin-induced currents with distinct desensitization and Ca2+ dependence in rat trigeminal ganglion cells

1996 ◽  
Vol 75 (4) ◽  
pp. 1503-1514 ◽  
Author(s):  
L. Liu ◽  
S. A. Simon
Keyword(s):  
Trigeminal Ganglion ◽  
Ganglion Cells ◽  
Reversal Potential ◽  
Outward Current ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Inward Currents ◽  
Current Densities ◽  
Rapid Current ◽  
Rate Of Activation

1. Whole cell patch-clamp records from cultured rat trigeminal ganglion cells having soma diameters ranging from 20 to 50 microM revealed that capsaicin activated two inward currents and an outward current. At -60 mV, the inward currents could be distinguished by their different peak times, which were 4.2 +/- 3.1 and 41.4 +/- 16.4 (SD) s. 2. Cells with the smallest soma diameters had the largest current densities. 3. The more rapidly activating current had a linear current-voltage relation and a reversal potential near 0 mV. 4. The more slowly activating current is not a Ca(2+)-activated Cl- current. 5. The peak of the rapid current (Ip)-capsaicin concentration (C) relationship was characterized by Ip/Ipmax = [1 + (C/Kd)n]-1, where n = 1.2 and the dissociation constant (Kd) = 0.68 microM. 6. The rapidly activating current was heterogeneous in regards to both its rate of activation and extent of desensitization. In cells bathed in buffer containing calcium and held at -60 mV, most of the capsaicin-activated currents desensitized. Removal of extracellular Ca2+ could reduce, eliminate, or have no effect on desensitization. 7. At positive holding potentials the currents very slowly desensitized, even in the presence of Ca2+. 8. Repeated 30-s applications of 1 microM capsaicin separated by 0.5, 2.5, and 5.5 min all induced tachyphylaxis. Tachyphylaxis decreased exponentially until the current remained approximately constant. Decreasing the time between capsaicin applications increased the extent of tachyphylaxis, whereas elimination of extracellular Ca2+ markedly reduced tachyphylaxis.

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 Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

2013 ◽  
Vol 141 (4) ◽  
pp. 493-497 ◽  
Author(s):  
Yanyan Geng ◽  
Xiaoyu Wang ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Negative Slope ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Linear Current ◽  
Joint Application ◽  
Current Voltage ◽  
Α Subunits

Large-conductance, voltage- and Ca2+-activated K+ (BK) channels display near linear current–voltage (I-V) plots for voltages between −100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca2+ and Mg2+. This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca2+ and Mg2+ in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus β1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus β1 subunits with joint application of 2.5 mM Ca2+ plus 2.5 mM Mg2+, as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca2+/Mg2+, as they can reduce conductance and induce negative slopes.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

scholarly journals Single-channel Ca2+ imaging implicates Aβ1–42 amyloid pores in Alzheimer’s disease pathology

2011 ◽  
Vol 195 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Angelo Demuro ◽  
Martin Smith ◽  
Ian Parker
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Ion Channels ◽  
Single Channel ◽  
Membrane Integrity ◽  
Cytosolic Calcium ◽  
Xenopus Laevis Oocytes ◽  
Permeable Membrane ◽  
Membrane Pores ◽  
Pathological Mechanism

Oligomeric forms of Aβ peptides are implicated in Alzheimer’s disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca2+) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca2+-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca2+ influx in Xenopus laevis oocytes. Aβ1–42 oligomers evoked single-channel Ca2+ fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (Po) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high Po, large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca2+-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

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