scholarly journals External barium block of Shaker potassium channels: evidence for two binding sites.

1995 ◽  
Vol 106 (6) ◽  
pp. 1069-1087 ◽  
Author(s):  
R S Hurst ◽  
R Latorre ◽  
L Toro ◽  
E Stefani
Keyword(s):  
Binding Sites ◽  
Single Channel ◽  
Slow Component ◽  
Fast Component ◽  
K Channel ◽  
Apparent Dissociation Constant ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Barium Block ◽  
Shaker Potassium Channels

External barium ions inhibit K+ currents of Xenopus oocytes expressing ShH4 delta 6-46, the non-inactivating deletion of the Shaker K+ channel. At the macroscopic level, Ba2+ block comprises both a fast and a slow component. The fast component is less sensitive to Ba2+ (apparent dissociation constant at 0 mV, K(0), approximately 19.1 mM) than the slow component and is also less voltage dependent (apparent electrical distance, delta, approximately 0.14). The slow component (K(0), approximately 9.4 mM, delta approximately 0.25) is relieved by outward K+ current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast component of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unblocking reaction. In contrast, the slow component appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (tau) produced by 2 mM Ba2+ is approximately 159 s (holding potential, HP = -90 mV). However, the blocking rate of this slow component is not a linear function of external Ba2+ and tends to saturate at higher concentrations. This is inconsistent with a simple bi-molecular blocking reaction. These features of external Ba2+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow component of block.

Permeation and block of dopamine-modulated potassium channels on rat striatal neurons by cesium and barium ions

1996 ◽  
Vol 76 (3) ◽  
pp. 1413-1422 ◽  
Author(s):  
Y. J. Lin ◽  
G. J. Greif ◽  
J. E. Freedman
Keyword(s):  
Dissociation Constant ◽  
Single Channel ◽  
Reversal Potential ◽  
Negative Slope ◽  
K Channel ◽  
Striatal Neurons ◽  
Apparent Dissociation Constant ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inwardly Rectifying

1. In cell-attached patch-clamp recordings from freshly dissociated rat caudate-putamen neurons, an 85-pS inwardly rectifying K+ channel, which was previously found to be modulated by D2-like dopamine receptors, was blocked by externally applied BaCl2 or CsCl. 2. At concentrations between 100 and 500 microM, Ba2+ blockade was voltage dependent, with a greater block at hyperpolarized voltages, in a manner consistent with blockade of the channel pore. Single-channel currents were flickery, with intervening periods of more complete blockade, and block appeared to be time dependent, with an estimated electrical distance of 0.24 and an apparent dissociation constant of 205 microM at 0 mV. 3. At concentrations between 1 and 3 mM, Cs+ blockade was similarly voltage dependent, but without periods of longer blockade, with an electrical distance of 0.81 and an apparent dissociation constant of 625 microM at 0 mV. Cs+ could also permeate this channel at voltages near the K+ reversal potential. The current-voltage relationship displayed an anomalous negative slope conductance, in a manner inconsistent with a single-ion pore. 4. Smaller-conductance, dopamine-insensitive channels were blocked more potently by both Ba2+ and Cs+ than was the 85-pS channel, reflecting differences between inwardly rectifying K+ channels mediating resting conductance and those mediating dopamine receptor responses in striatal neurons.

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 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.

Internal and external TEA block in single cloned K+ channels

1991 ◽  
Vol 261 (4) ◽  
pp. C583-C590 ◽  
Author(s):  
G. E. Kirsch ◽  
M. Taglialatela ◽  
A. M. Brown
Keyword(s):  
Single Channel ◽  
Channel Structure ◽  
K Channel ◽  
Cdna Clones ◽  
K Channels ◽  
Open Time ◽  
Voltage Dependent ◽  
Internal Site ◽  
Channel Open Time ◽  
Single Channel Currents

Tetraethylammonium (TEA) has been used recently to probe natural and mutational variants of voltage-dependent K+ channels encoded by cDNA clones. Its usefulness as a probe of channel structure prompted us to examine the molecular mechanism by which TEA blocks single-channel currents in Xenopus oocytes expressing the rat brain K+ channel, RCK2. TEA at the intracellular surface of membrane patches decreased channel open time and increased the duration of closed intervals. Tetrapentylammonium had similar but more potent effects. Extracellular application of TEA caused an apparent reduction of single-channel amplitude. Block was slower at the high-affinity internal site than at the low-affinity external site. Internal TEA selectively blocks open K+ channels, and the voltage dependence of the block indicates that the binding site lies within the membrane electric field at a point 25% of the distance from the cytoplasmic margin. External TEA also interacts with the open channel but is less sensitive to membrane potential. The results indicate that the internal and external TEA binding sites define the inner and outer margins of the aqueous pore.

scholarly journals Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.

1987 ◽  
Vol 90 (1) ◽  
pp. 27-47 ◽  
Author(s):  
A Hermann ◽  
C Erxleben
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Pacemaker Activity ◽  
Dependent Manner ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
External Application ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

scholarly journals A conductance maximum observed in an inward-rectifier potassium channel.

1994 ◽  
Vol 104 (3) ◽  
pp. 477-486 ◽  
Author(s):  
Z Lu ◽  
R MacKinnon
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Inward Rectifier ◽  
Ion Concentration ◽  
K Channel ◽  
Single Channels ◽  
Ligand Interaction ◽  
Membrane Biology ◽  
Maximum Value ◽  
Conduction Pathway

One prediction of a multi-ion pore is that its conductance should reach a maximum and then begin to decrease as the concentration of permeant ion is raised equally on both sides of the membrane. A conductance maximum has been observed at the single-channel level in gramicidin and in a Ca(2+)-activated K+ channel at extremely high ion concentration (> 1,000 mM) (Hladky, S. B., and D. A. Haydon. 1972. Biochimica et Biophysica Acta. 274:294-312; Eisenmam, G., J. Sandblom, and E. Neher. 1977. In Metal Ligand Interaction in Organic Chemistry and Biochemistry. 1-36; Finkelstein, P., and O. S. Andersen. 1981. Journal of Membrane Biology. 59:155-171; Villarroel, A., O. Alvarez, and G. Eisenman. 1988. Biophysical Journal. 53:259a. [Abstr.]). In the present study we examine the conductance-concentration relationship in an inward-rectifier K+ channel, ROMK1. Single channels, expressed in Xenopus oocytes, were studied using inside-out patch recording in the absence of internal Mg2+ to eliminate blockade of outward current. Potassium, at equal concentrations on both sides of the membrane, was varied from 10 to 1,000 mM. As K+ was raised from 10 mM, the conductance increased steeply and reached a maximum value (39 pS) at 300 mM. The single-channel conductance then became progressively smaller as K+ was raised beyond 300 mM. At 1000 mM K+, the conductance was reduced to approximately 75% of its maximum value. The shape of the conductance-concentration curve observed in the ROMK1 channel implies that it has multiple K(+)-occupied binding sites in its conduction pathway.

scholarly journals Shaker potassium channel gating. I: Transitions near the open state.

1994 ◽  
Vol 103 (2) ◽  
pp. 249-278 ◽  
Author(s):  
T Hoshi ◽  
W N Zagotta ◽  
R W Aldrich
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Activation Process ◽  
Time Data ◽  
Shaker Potassium Channel ◽  
Voltage Dependent ◽  
Average Current ◽  
Shaker Potassium Channels ◽  
Kinetics Of ◽  
Deactivation Process

Kinetics of single voltage-dependent Shaker potassium channels expressed in Xenopus oocytes were studied in the absence of fast N-type inactivation. Comparison of the single-channel first latency distribution and the time course of the ensemble average current showed that the activation time course and its voltage dependence are largely determined by the transitions before first opening. The open dwell time data are consistent with a single kinetically distinguishable open state. Once the channel opens, it can enter at least two closed states which are not traversed frequently during the activation process. The rate constants for the transitions among these closed states and the open state are nearly voltage-independent at depolarized voltages (> -30 mV). During the deactivation process at more negative voltages, the channel can close directly to a closed state in the activation pathway in a voltage-dependent fashion.

scholarly journals Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.

1988 ◽  
Vol 91 (3) ◽  
pp. 335-349 ◽  
Author(s):  
R MacKinnon ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
External Solution ◽  
Dissociation Rate ◽  
K Channel ◽  
Internal Solution ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
K Concentration ◽  
A Site ◽  
Low K

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

scholarly journals Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel.

1987 ◽  
Vol 90 (3) ◽  
pp. 427-449 ◽  
Author(s):  
C Miller ◽  
R Latorre ◽  
I Reisin
Keyword(s):  
Lipid Bilayers ◽  
Dissociation Rate ◽  
K Channel ◽  
Single Channels ◽  
Closed Channel ◽  
Association Rate ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
High Conductance

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.

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