Interactive domains between pore loops of the yeast K+ channel TOK1 associate with extracellular K+ sensitivity

Gating of the outward-rectifying K+ channel TOK1 of Saccharomyces cerevisiae is controlled by membrane voltage and extracellular K+ concentration. Previous studies identified two kinetically distinct effects of K+, and site-mutagenic analysis associated these K+-dependencies with domains of the extracellular turrets of the channel protein. We have mapped the TOK1 pore domains to extant K+ channel crystal structures to target additional residues contributing to TOK1 gating. Leu270, located in the first pore domain of TOK1, was found to be critical for gating and its K+ sensitivity. Analysis of amino acid substitutions indicated that spatial position of the polypeptide backbone is a primary factor determining gating sensitivity to K+. The strongest effects, with L270Y, L270F and L270W, led to more than a 30-fold decrease in apparent K+ affinity and an inversion in the apparent K+-dependence of voltage-dependent gating compared with the wild-type current. A partial rescue of wild-type gating was obtained on substitution in the second pore domain with the double mutant L270D/A428Y. These, and additional results, demarcate extracellular domains that are associated with the K+-sensitivity of TOK1 and they offer primary evidence for a synergy in gating between the two pore domains of TOK1, demonstrating an unexpected degree of long-distance interaction across the mouth of the K+ channel.

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Isolation and Functional Determination of SKOR Potassium Channel in Purple Osier Willow, Salix purpurea

International Journal of Genomics ◽

10.1155/2021/6669509 ◽

2021 ◽

Vol 2021 ◽

pp. 1-7

Author(s):

Yahui Chen ◽

Xuefeng Peng ◽

Jijie Cui ◽

Hongxia Zhang ◽

Jiang Jiang ◽

...

Keyword(s):

Patch Clamp ◽

Membrane Voltage ◽

K Channel ◽

Long Distance ◽

Type K ◽

Voltage Dependent ◽

Salix Purpurea ◽

K Concentration ◽

Electrophysiological System ◽

Forest Plants

Potassium (K+) plays key roles in plant growth and development. However, molecular mechanism studies of K+ nutrition in forest plants are largely rare. In plants, SKOR gene encodes for the outward rectifying Shaker-type K+ channel that is responsible for the long-distance transportation of K+ through xylem in roots. In this study, we determined a Shaker-type K+ channel gene in purple osier (Salix purpurea), designated as SpuSKOR, and determined its function using a patch clamp electrophysiological system. SpuSKOR was closely clustered with poplar PtrSKOR in the phylogenetic tree. Quantitative real-time PCR (qRT-PCR) analyses demonstrated that SpuSKOR was predominantly expressed in roots, and expression decreased under K+ depletion conditions. Patch clamp analysis via HEK293-T cells demonstrated that the activity of the SpuSKOR channel was activated when the cell membrane voltage reached at -10 mV, and the channel activity was enhanced along with the increase of membrane voltage. Outward currents were recorded and induced in response to the decrease of external K+ concentration. Our results indicate that SpuSKOR is a typical voltage dependent outwardly rectifying K+ channel in purple osier. This study provides theoretical basis for revealing the mechanism of K+ transport and distribution in woody plants.

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

The Journal of General Physiology ◽

10.1085/jgp.91.3.335 ◽

1988 ◽

Vol 91 (3) ◽

pp. 335-349 ◽

Cited By ~ 250

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.

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Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel

eLife ◽

10.7554/elife.00594 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 122

Author(s):

Anirban Banerjee ◽

Alice Lee ◽

Ernest Campbell ◽

Roderick MacKinnon

Keyword(s):

Electron Density ◽

Selectivity Filter ◽

K Channel ◽

Wild Type ◽

Kv Channel ◽

Pore Blocking ◽

Kv Channels ◽

Filter Structure ◽

Conduction Pathway ◽

Voltage Dependent

Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter.

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AKT2/3 Subunits Render Guard Cell K+ Channels Ca2+ Sensitive

The Journal of General Physiology ◽

10.1085/jgp.200409211 ◽

2005 ◽

Vol 125 (5) ◽

pp. 483-492 ◽

Cited By ~ 45

Author(s):

Natalya Ivashikina ◽

Rosalia Deeken ◽

Susanne Fischer ◽

Peter Ache ◽

Rainer Hedrich

Keyword(s):

Guard Cell ◽

Guard Cells ◽

Inward Rectifier ◽

Hek293 Cells ◽

K Channel ◽

Dependent Manner ◽

Wild Type ◽

K Channels ◽

Isolation And Characterization ◽

Voltage Dependent

Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel.

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Dynamic Interaction of S5 and S6 during Voltage-Controlled Gating in a Potassium Channel

The Journal of General Physiology ◽

10.1085/jgp.118.2.157 ◽

2001 ◽

Vol 118 (2) ◽

pp. 157-170 ◽

Cited By ~ 19

Author(s):

Felipe Espinosa ◽

Richard Fleischhauer ◽

Anne McMahon ◽

Rolf H. Joho

Keyword(s):

Single Channel ◽

Side Chain ◽

State Transitions ◽

K Channel ◽

Side Chains ◽

Wild Type ◽

Closed State ◽

Voltage Dependent ◽

Bulky Side Chain ◽

Small Side

A gain-of-function mutation in the Caenorhabditis elegans exp-2 K+-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501–2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at −160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K+ channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73–78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K+ ions at potentials as negative as −120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a “two-gate model” with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654–661).

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Accumulation of inactivation in a cloned transient K+ channel (AKv1.1a) of Aplysia

Journal of Neurophysiology ◽

10.1152/jn.1995.74.3.1248 ◽

1995 ◽

Vol 74 (3) ◽

pp. 1248-1257 ◽

Cited By ~ 5

Author(s):

Y. Furukawa

Keyword(s):

Short Interval ◽

Low Frequency ◽

K Channel ◽

Amino Terminal ◽

Recovery From Inactivation ◽

Voltage Dependent ◽

Inactivation Gating ◽

A Cell ◽

K Concentration ◽

External K

1. Inactivation of a cloned Aplysia K+ channel, AKv1.1a, expressed in Xenopus oocytes was examined by a cell-attached macropatch recording. A fast macroscopic inactivation (the time constant for decay was in the range of 20-40 ms) in response to a depolarizing command pulse was insensitive to the concentration of external K+ (2-100 mM KCl). 2. By contrast, recovery from inactivation was extremely slow and dependent on external K+. When the concentration of external KCl was 2-3 mM, a patched membrane had to be held at hyperpolarized potential for > 40 s for a full recovery. The recovery was greatly accelerated if external K+ concentration was increased. A tail current following a command pulse long enough to inactivate most of the channels showed a marked rising phase. 3. A consequence of the slow recovery from inactivation was that AKv1.1a showed a marked accumulation of the inactivation following repetitive pulses, even at low frequency (< 0.1 Hz). When two depolarizing pulses were applied at a short interval, the current during a second pulse was smaller than the current at the end of the preceding pulse. This is a phenomenon called "cumulative inactivation." The onset and the extent of cumulative inactivation of AKv1.1a were voltage dependent but relatively insensitive to external K+ concentration. An amino terminal deletion mutant of AKv1.1a that lacks the fast N-type inactivation did not show cumulative inactivation. 4. These results suggest that the inactivation gating by the amino terminal region of AKv1.1a has a similarity to open-channel blockade, and that the cumulative inactivation can also be dependent on the amino terminal cytoplasmic domain of K+ channels.

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