Surface Expression and Single Channel Properties of KCNQ2/KCNQ3, M-type K+Channels Involved in Epilepsy

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.

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Single-channel properties and regulation of pinacidil/glibenclamide-sensitive K+ channels in follicular cells from Xenopus oocyte

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf00374601 ◽

1993 ◽

Vol 424 (2) ◽

pp. 113-121 ◽

Cited By ~ 30

Author(s):

Eric Honor� ◽

Michel Lazdunski

Keyword(s):

Xenopus Oocyte ◽

Single Channel ◽

Follicular Cells ◽

K Channels ◽

Channel Properties

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

The Journal of General Physiology ◽

10.1085/jgp.111.2.363 ◽

1998 ◽

Vol 111 (2) ◽

pp. 363-379 ◽

Cited By ~ 11

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).

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Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

The Journal of General Physiology ◽

10.1085/jgp.200509457 ◽

2006 ◽

Vol 127 (2) ◽

pp. 159-169 ◽

Cited By ~ 29

Author(s):

Jill Thompson ◽

Ted Begenisich

Keyword(s):

Single Channel ◽

Expression System ◽

Chemical Activation ◽

K Channel ◽

Channel Activity ◽

Single Family ◽

Open Probability ◽

K Channels ◽

Channel Proteins ◽

K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

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Pseudomonas fluorescens lipopolysaccharide inhibits both delayed rectifier and transient A-type K+ channels of cultured rat cerebellar granule neurons

Brain Research ◽

10.1016/s0006-8993(03)03055-5 ◽

2003 ◽

Vol 983 (1-2) ◽

pp. 185-192 ◽

Cited By ~ 6

Author(s):

Sana Mezghani-Abdelmoula ◽

Sylvie Chevalier ◽

Olivier Lesouhaitier ◽

Nicole Orange ◽

Marc G.J. Feuilloley ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Cerebellar Granule Neurons ◽

Delayed Rectifier ◽

Granule Neurons ◽

Cerebellar Granule ◽

K Channels ◽

Type K

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cAMP-dependent regulation of IKs single-channel kinetics

The Journal of General Physiology ◽

10.1085/jgp.201611734 ◽

2017 ◽

Vol 149 (8) ◽

pp. 781-798 ◽

Cited By ~ 10

Author(s):

Emely Thompson ◽

Jodene Eldstrom ◽

Maartje Westhoff ◽

Donald McAfee ◽

Elise Balse ◽

...

Keyword(s):

Action Potential ◽

Single Channel ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Surface Expression ◽

Voltage Sensor ◽

Single Channels ◽

Adrenergic Stimulation ◽

Channel Opening ◽

Sensor Activation

The delayed potassium rectifier current, IKs, is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3′-5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance–voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.

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