scholarly journals Revisiting inward rectification: K ions permeate through Kir2.1 channels during high-affinity block by spermidine

2012 ◽  
Vol 139 (3) ◽  
pp. 245-259 ◽  
Author(s):  
Tai-An Liu ◽  
Hsueh-Kai Chang ◽  
Ru-Chi Shieh
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Inward Rectification ◽  
Excitable Cells ◽  
High Affinity ◽  
Current State ◽  
Molecular Events ◽  
Zero Current ◽  
Outward Currents ◽  
Voltage Dependent

Outward currents through Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells, and such currents are subjected to voltage-dependent block by intracellular Mg2+ and polyamines that bind to both high- and low-affinity sites on the channels. Under physiological conditions, high-affinity block is saturated and yet outward Kir2.1 currents can still occur, implying that high-affinity polyamine block cannot completely eliminate outward Kir2.1 currents. However, the underlying molecular mechanism remains unknown. Here, we show that high-affinity spermidine block, rather than completely occluding the single-channel pore, induces a subconducting state in which conductance is 20% that of the fully open channel. In a D172N mutant lacking the high-affinity polyamine-binding site, spermidine does not induce such a substate. However, the kinetics for the transitions between the substate and zero-current state in wild-type channels is the same as that of low-affinity block in the D172N mutant, supporting the notion that these are identical molecular events. Thus, the residual outward current after high-affinity spermidine block is susceptible to low-affinity block, which determines the final amplitude of the outward current. This study provides a detailed insight into the mechanism underlying the emergence of outward Kir2.1 currents regulated by inward rectification attributed to high- and low-affinity polyamine blocks.

scholarly journals Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander.

1990 ◽  
Vol 96 (4) ◽  
pp. 809-834 ◽  
Author(s):  
K Sugimoto ◽  
J H Teeter
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Tiger Salamander ◽  
Inward Current ◽  
Receptor Cells ◽  
Outward Currents ◽  
Taste Cells ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
K Currents

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca-Induced K+-Outward Current in Paramecium Tetraurelia

1980 ◽  
Vol 88 (1) ◽  
pp. 293-304 ◽  
Author(s):  
YOUKO SATOW ◽  
CHING KUNG
Keyword(s):  
Steady State ◽  
Voltage Clamp ◽  
Outward Current ◽  
Paramecium Tetraurelia ◽  
Ca Channels ◽  
Outward Currents ◽  
Ciliary Reversal ◽  
Voltage Dependent ◽  
K Current ◽  
K Outward Current

Late K-outward currents upon membrane depolarization were recorded in Paramecium tetraurelia under a voltage clamp. A Ca-induced K-outward component is demonstrated by subtracting the value of the outward current in a pawn A mutant lacking functional Ca-channels (pwA500). The Ca-induced K-outward current activates slowly, reaching a peak after 100 to 1000 ms. The current then remains steady or reaches the steady state after a decline of several seconds. EGTA2- injection experiments show that the Ca-induced K-outward current is dependent on the internal Ca2+ concentration. The current is shown to depend on the voltage-dependent Ca conductance, by study of the leaky pawn A mutant (pwA132), which has a lowered Ca conductance as well as a lowered Ca-induced K-current. The Ca-induced GK is thus indirectly dependent on the voltage. The maximal GK is about 40 nmho/cell at + 7 mV in 4 mM-K+. The Ca-induced K current is sustained throughout the prolonged depolarization and the prolonged ciliary reversal.

scholarly journals Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

1997 ◽  
Vol 273 (6) ◽  
pp. C2010-C2021 ◽  
Author(s):  
S. D. Koh ◽  
G. M. Dick ◽  
K. M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Single Channel ◽  
Outward Current ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Murine Colon ◽  
Small Conductance

The patch-clamp technique was used to determine the ionic conductances activated by ATP in murine colonic smooth muscle cells. Extracellular ATP, UTP, and 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased outward currents in cells with amphotericin B-perforated patches. ATP (0.5–1 mM) did not affect whole cell currents of cells dialyzed with solutions containing ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. Apamin (3 × 10−7M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and 2-MeS-ATP increased the open probability of small-conductance, Ca2+-dependent K+ channels with a slope conductance of 5.3 ± 0.02 pS. Caffeine (500 μM) enhanced the open probability of the small-conductance K+ channels, and ATP had no effect after caffeine. Pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS, 10−4 M), a nonselective P2 receptor antagonist, prevented the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had no effect on the response to caffeine. ATP-induced hyperpolarization in the murine colon may be mediated by P2y-induced release of Ca2+ from intracellular stores and activation of the 5.3-pS Ca2+-activated K+ channels.

scholarly journals Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2020 ◽  
Vol 21 (14) ◽  
pp. 4876
Author(s):  
Zbigniew Burdach ◽  
Agnieszka Siemieniuk ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Inward Current ◽  
Bath Solution ◽  
Outward Currents ◽  
Inward Currents

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells

1987 ◽  
Vol 253 (1) ◽  
pp. H210-H214
Author(s):  
M. Horie ◽  
H. Irisawa
Keyword(s):  
Guinea Pig ◽  
Action Potentials ◽  
Single Channel ◽  
Outward Current ◽  
Atrial Cells ◽  
Voltage Dependent ◽  
K Current ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Low K

Rectifying properties of the acetylcholine (ACh)-sensitive K+ channels were studied using a patch-clamp method in single atrial cells prepared enzymatically from adult guinea pig hearts. In the presence of micromolar concentration of nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue 5'-guanylylimidodiphosphate (GppNHp) and the absence of Mg2+ at the inner surface of patch membrane [( Mg2+]i), the channel activity recovered in inside-out patch condition. The single channel conductance became ohmic between -80 and +80 mV (symmetrical 150 mM K+ solutions). The rapid relaxation of outward single channel currents was disclosed on a depolarization. [Mg2+]i blocked the outward current through the channel dose- and voltage-dependently and also induced a dose-dependent increase in the channel activation. The apparent paradoxical role of [Mg2+]i is important for the cholinergic control in the heart; voltage-dependent Mg block ensures a low K+ conductance of cell membrane at the plateau of action potentials during the exposure to ACh, thereby slowing the heart rate without unfavorable shortening of the action potentials.

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.

Transient potassium currents in avian sensory neurons

1990 ◽  
Vol 63 (4) ◽  
pp. 725-737 ◽  
Author(s):  
S. K. Florio ◽  
C. D. Westbrook ◽  
M. R. Vasko ◽  
R. J. Bauer ◽  
J. L. Kenyon
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Potassium Currents ◽  
Delayed Rectifier ◽  
Transient Outward Current ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Small Component ◽  
Whole Cell ◽  
Outward Currents

1. We used the patch-clamp technique to study voltage-activated transient potassium currents in freshly dispersed and cultured chick dorsal root ganglion (DRG) cells. Whole-cell and cell-attached patch currents were recorded under conditions appropriate for recording potassium currents. 2. In whole-cell experiments, 100-ms depolarizations from normal resting potentials (-50 to -70 mV) elicited sustained outward currents that inactivated over a time scale of seconds. We attribute this behavior to a component of delayed rectifier current. After conditioning hyperpolarizations to potentials negative to -80 mV, depolarizations elicited transient outward current components that inactivated with time constants in the range of 8-26 ms. We attribute this behavior to a transient outward current component. 3. Conditioning hyperpolarizations increased the rate of activation of the net outward current implying that the removal of inactivation of the transient outward current allows it to contribute to early outward current during depolarizations from negative potentials. 4. Transient current was more prominent on the day the cells were dispersed and decreased with time in culture. 5. In cell-attached patches, single channels mediating outward currents were observed that were inactive at resting potentials but were active transiently during depolarizations to potentials positive to -30 mV. The probability of channels being open increased rapidly (peaking within approximately 6 ms) and then declined with a time constant in the range of 13-30 ms. With sodium as the main extracellular cation, single-channel conductances ranged from 18 to 32 pS. With potassium as the main extracellular cation, the single-channel conductance was approximately 43 pS, and the channel current reversed near 0 mV, as expected for a potassium current. 6. We conclude that the transient potassium channels mediate the component of transient outward current seen in the whole-cell experiments. This current is a relatively small component of the net current during depolarizations from normal resting potentials, but it can contribute significant outward current early in depolarizations from hyperpolarized potentials.

scholarly journals Ring of Negative Charge in BK Channels Facilitates Block by Intracellular Mg2+ and Polyamines through Electrostatics

2006 ◽  
Vol 128 (2) ◽  
pp. 185-202 ◽  
Author(s):  
Yaxia Zhang ◽  
Xiaowei Niu ◽  
Tinatin I. Brelidze ◽  
Karl L. Magleby
Keyword(s):  
Negative Charge ◽  
Outward Current ◽  
Bk Channels ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Conduction Pathway ◽  
Boltzmann Function ◽  
Voltage Dependent ◽  
Hill Coefficients ◽  
The Hill

Intracellular Mg2+ and natural polyamines block outward currents in BK channels in a highly voltage-dependent manner. Here we investigate the contribution of the ring of eight negatively charged residues (4 x E321/E324) at the entrance to the inner vestibule of BK channels to this block. Channels with or without (E321N/E324N) the ring of negative charge were expressed in oocytes and unitary currents were recorded from inside-out patches over a range of intracellular Mg2+ and polyamine concentrations. Removing the ring of charge greatly decreased the block, increasing KBap (0 mV) for Mg2+ block from 48.3 ± 3.0 to 143 ± 8 mM, and for spermine block from 8.0 ± 1.0 to 721 ± 9 mM (150 mM symmetrical KCl). Polyamines with fewer amine groups blocked less: putrescine < spermidine < spermine. An equation that combined an empirical Hill function for block together with a Boltzmann function for the voltage dependence of KBap described the voltage and concentration dependence of the block for channels with and without the ring of charge. The Hill coefficients for these descriptions were <1 for both Mg2+ and spermine block, and were unchanged by removing the ring of charge. When KCli was increased from 150 mM to 3 M, the ring of charge no longer facilitated block, Mg2+ block was reduced, spermine block became negligible, and the Hill coefficients became ∼1.0. BK channels in cell-attached oocyte patches displayed inward rectification, which was reduced for channels without the ring of charge. Taken together, these observations suggest that the ring of negative charge facilitates block through a preferential electrostatic attraction of Mg2+ and polyamine over K+. This preferential attraction of multivalent blockers over monovalent K+ would decrease the K+ available at the inner vestibule to carry outward current in the presence of Mg2+ or polyamines, while increasing the concentration of blocker available to enter and block the conduction pathway.

Segment-specific effects of FMRFamide on membrane properties of heart interneurons in the leech

1995 ◽  
Vol 74 (4) ◽  
pp. 1485-1497 ◽  
Author(s):  
J. Schmidt ◽  
S. Gramoll ◽  
R. L. Calabrese
Keyword(s):  
Outward Current ◽  
Membrane Conductance ◽  
Membrane Properties ◽  
Inward Current ◽  
Specific Effects ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents ◽  
K Current ◽  
Conductance Increase

1. The effects of Phe-Met-Arg-Phe (FMRF)amide (10(-6) M) on membrane properties of heart interneurons in the third, fourth, and fifth segmental ganglia [HN(3), HN(4), and HN(5) cells, respectively] of the leech were studied using discontinuous current-clamp and single-electrode voltage-clamp techniques. FMRFamide was focally applied onto the soma of the cell under investigation. 2. Application of FMRFamide depolarized HN(3) and HN(4) cells by evoking an inward current. These responses were subject to pronounced desensitization. The inward currents evoked by application of FMRFamide were associated with an increase in membrane conductance and appeared to be voltage dependent. Currents were enhanced at more depolarized potentials. 3. The responsiveness of the HN(3) and HN(4) cells was not affected when the Ca2+ concentration in the bath saline was reduced from normal (1.8 mM) to 0.1 mM. The depolarizing response on application of FMRFamide was blocked when Co2+ was substituted for Ca2+. 4. HN(3) and HN(4) cells did not respond to FMRFamide application in Na(+)-free solution. Inward currents were largely reduced when bath saline with 30% of the normal Na+ concentration was used. When Li+ was substituted for Na+ in the saline, application of FMRFamide still evoked depolarizing responses in HN(3) and HN(4) cells. 5. We conclude that focal application of FMRFamide onto the somata of HN(3) and HN(4) cells evokes a voltage-dependent inward current, carried largely by Na+. 6. Focal application of FMRFamide onto somata of HN(5) cells hyperpolarized these cells by activating a voltage-dependent outward current. 7. HN(5) cells were loaded with Cl- until inhibitory postsynaptic potentials carried by Cl- reversed. Cl(-)-loaded cells still responded with a hyperpolarization when FMRFamide was applied onto their somata. Therefore the outward current evoked by FMRFamide appears to be mediated by a K+ conductance increase. 8. Application of FMRFamide onto the somata of HN(5) cells enhanced outward currents that were evoked by depolarizing voltage steps from a holding potential of -45 mV. 9. We conclude that the hyperpolarizing response of HN(5) cells to focal application of FMRFamide onto their somata is the result of an up-regulation of a voltage-dependent K+ current.

scholarly journals Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

1992 ◽  
Vol 100 (5) ◽  
pp. 783-801 ◽  
Author(s):  
L W Haynes
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Single Channel ◽  
Inward Rectification ◽  
Pipette Solution ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Extracellular Side ◽  
Current Voltage Relation ◽  
Cytoplasmic Side

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

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