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 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.

Ca2+ inhibits outward current through single K+ channels in canine atrial myocyte sarcolemma

1988 ◽  
Vol 254 (3) ◽  
pp. C397-C403 ◽  
Author(s):  
J. K. Bubien ◽  
H. Van Der Heyde ◽  
W. T. Woods
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Transient Outward Current ◽  
Single Channels ◽  
Bathing Solution ◽  
Voltage Sensitivity ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Single Channel Currents

Single-channel currents in canine atrial cells were recorded by the patch-clamp technique in a bathing solution containing 150 mM [K+] and pipette solutions containing 5 mM [K+]. One kind of current was observed in 56% of 178 cell-free patches and in 3% of 60 patches in the cell-attached configuration. Single-channel amplitude varied in direct proportion to the bath [K+]. Openings of these single channels were prevented when bath [Ca2+] exceeded 1 microM. Below this concentration single-channel percent open time was inversely proportional to log [Ca2+]. Inward current was observed at hyperpolarized membrane potentials in some patches. There was no apparent steady-state voltage sensitivity. These properties suggest that the K+ channel described in this study (gK+LF), a low transition frequency K+ conductor, may be distinct from single K+ channels previously studied in cardiac myocyte sarcolemmae. The single-channel response to "intracellular" free [Ca2+] and the single-channel kinetic characteristics described in this study are similar to the macroscopic "long-lasting transient outward current" (IIO) described by Escande et al. [Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H142-H148, 1987] in human atrial myocytes (tau open = 29.6 ms, tau inactivation = 35.7 ms, respectively). This suggests that gK+LF channels may carry IIO.

scholarly journals Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.

1994 ◽  
Vol 103 (4) ◽  
pp. 583-604 ◽  
Author(s):  
V Urbach ◽  
E van Kerkhove ◽  
B J Harvey
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Ringer Solution ◽  
Inward Rectifier ◽  
Inward Rectification ◽  
K Channel ◽  
Intact Cells ◽  
Principal Cells ◽  
Kir Channel ◽  
Current Voltage

Inward-rectifier K channel: using macroscopic voltage clamp and single-channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current-voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell-attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward-rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.

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 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.

Ca2+-activated K+ channels in cultured medullary thick ascending limb cells

1987 ◽  
Vol 252 (2) ◽  
pp. C121-C127 ◽  
Author(s):  
S. E. Guggino ◽  
W. B. Guggino ◽  
N. Green ◽  
B. Sacktor
Keyword(s):  
Cell Membrane ◽  
Single Channel ◽  
Apical Cell ◽  
Outward Current ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Patch Clamp Technique ◽  
Apical Cell Membrane ◽  
K Channels ◽  
Medullary Thick Ascending Limb

The conductive properties of a clone of medullary thick ascending limb (MTAL) cells (GRB-MAL1) were assessed using conventional microelectrodes and the patch clamp technique. The apical cell membrane potential (Va) of MTAL cells was -46 +/- 3 mV. Addition of Ba2+ (1 mM) to the apical solution induced a 22 +/- 2 mV depolarization of Va, whereas furosemide hyperpolarized Va by -5 +/- 1 mV. In the cell-attached patch configuration, the most frequently occurring channel had a single channel conductance of 121 +/- 5 pS and carried outward current. In excised patches, current movement was down the electrochemical K+ gradient. Fluctuations were activated by depolarization of Va and by increasing Ca2+ concentration on the intracellular face. Micromolar amounts of Ba2+ on the intracellular face of the membrane inhibited channel activity. We conclude that cultures of MTAL cells GRB-MAL1 retain at least two of the properties of the mature phenotype, namely, an apical K+ conductance and a sensitivity to loop diuretics; the most frequently occurring channel in the apical cell membrane is a Ca2+-activated, maxi-K+ channel; and, finally Ca2+-activated K+ channels may play a role in generating the apical K+ conductance in cultured MTAL cells.

scholarly journals Ionic interactions of Ba2+ blockades in the MthK K+ channel

2014 ◽  
Vol 144 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Rui Guo ◽  
Weizhong Zeng ◽  
Hengjun Cui ◽  
Liping Chen ◽  
Sheng Ye
Keyword(s):  
Single Channel ◽  
K Channel ◽  
K Channels ◽  
X Ray ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Single File ◽  
Conduction Pathway ◽  
Internal Side ◽  
Channel Properties

The movement and interaction of multiple ions passing through in single file underlie various fundamental K+ channel properties, from the effective conduction of K+ ions to channel blockade by Ba2+ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba2+ with permeant ions within the ion conduction pathway of the MthK K+ channel. We found that, as typical of K+ channels, the MthK channel was blocked by Ba2+ at the internal side, and the Ba2+-blocking effect was enhanced by external K+. We also obtained crystal structures of the MthK K+ channel pore in both Ba2+–Na+ and Ba2+–K+ environments. In the Ba2+–Na+ environment, we found that a single Ba2+ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba2+–K+ environment, Ba2+ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba2+ blockade of K+ channels.

scholarly journals Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+ -activated K+ channel.

1988 ◽  
Vol 92 (5) ◽  
pp. 569-586 ◽  
Author(s):  
J Neyton ◽  
C Miller
Keyword(s):  
External Solution ◽  
K Channel ◽  
Enhancement Effect ◽  
Single Channels ◽  
Internal Solution ◽  
Conduction Pathway ◽  
Lock In Effect ◽  
Lock In ◽  
High Conductance ◽  
External K

In this study, high-conductance Ca2+-activated K+ channels from rat skeletal muscle were incorporated into planar phospholipid bilayers, and discrete blockade of single channels by Ba2+ was studied. With 150 mM K+ held constant in the internal solution, increasing external K+ over the range 100-1,000 mM raises the rate of Ba2+ dissociation. This "enhancement effect," which operates at K+ concentrations 3-4 orders of magnitude higher than those required for the "lockin" effect described previously, depends on applied voltage, saturates with K+ concentration, and is not observed with Na+. The voltage dependence of the Ba2+ off-rate varies with external K+ in a way suggesting that K+, entering the channel from the external side, forces Ba2+ dissociation to the internal solution. With K+ held fixed in the external solution, the Ba2+ off-rate decreases as internal K+ is raised over the range 0-50 mM. This "lock-in" effect is similar to that seen on the external side (Neyton and Miller, 1988), except that the internal lock-in site is of lower affinity and shows only a fivefold preference for K+ over Na+. All the results taken together argue strongly that this channel's conduction pathway contains four sites of very high affinity for K+, all of which may be simultaneously occupied under normal conducting conditions. According to this view, the mutual destabilization resulting from this high ionic occupancy leads to the unusually high conductance of this K+-specific channel.

scholarly journals Control of Single Channel Conductance in the Outer Vestibule of the Kv2.1 Potassium Channel

2006 ◽  
Vol 128 (2) ◽  
pp. 231-246 ◽  
Author(s):  
Josef G. Trapani ◽  
Payam Andalib ◽  
Joseph F. Consiglio ◽  
Stephen J. Korn
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Markov Modeling ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Current Magnitude ◽  
Hidden Markov Modeling ◽  
Outer Vestibule ◽  
Dependent Change ◽  
Conduction Pathway

Current magnitude in Kv2.1 potassium channels is modulated by external [K+]. In contrast to behavior expected from the change in electrochemical driving force, outward current through Kv2.1 channels becomes larger when extracellular [K+] is increased within the physiological range. The mechanism that underlies this unusual property involves the opening of Kv2.1 channels into one of two different outer vestibule conformations, which are defined by their sensitivity to TEA. Channels that open into a TEA-sensitive conformation generate larger macroscopic currents, whereas channels that open into a TEA-insensitive conformation generate smaller macroscopic currents. At higher [K+], more channels open into the TEA-sensitive conformation. In this manuscript, we examined the mechanism by which the conformational change produced a change in current magnitude. We started by testing the simplest hypothesis: that each pharmacologically defined channel conformation produces a different single channel conductance, one smaller and one larger, and that the [K+]-dependent change in current magnitude reflects the [K+]-dependent change in the percentage of channels that open into each of the two conformations. Using single channel and macroscopic recordings, as well as hidden Markov modeling, we were able to quantitatively account for [K+]-dependent regulation of macroscopic current with this model. Combined with previously published work, these results support a model whereby an outer vestibule lysine interferes with K+ flux through the channel, and that the [K+]-dependent change in orientation of this lysine alters single channel conductance by changing the level of this interference. Moreover, these results provide an experimental example of single channel conductance being modulated at the outer end of the conduction pathway by a mechanism that involves channel activation into open states with different outer vestibule conformations.

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