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

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

Biochemical Journal ◽

10.1042/bj20051380 ◽

2006 ◽

Vol 393 (3) ◽

pp. 645-655 ◽

Cited By ~ 9

Author(s):

Ingela Johansson ◽

Michael R. Blatt

Keyword(s):

Double Mutant ◽

K Channel ◽

Wild Type ◽

Long Distance ◽

Voltage Dependent ◽

Polypeptide Backbone ◽

K Concentration ◽

Pore Domain ◽

Distance Interaction ◽

Pore Domains

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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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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Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.

The Journal of General Physiology ◽

10.1085/jgp.95.5.791 ◽

1990 ◽

Vol 95 (5) ◽

pp. 791-818 ◽

Cited By ~ 21

Author(s):

Y Segal ◽

L Reuss

Keyword(s):

Patch Clamp ◽

Apical Membrane ◽

Membrane Conductance ◽

Membrane Depolarization ◽

Membrane Voltage ◽

K Channel ◽

Gallbladder Epithelium ◽

Patch Clamp Technique ◽

K Channels ◽

Necturus Gallbladder

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.

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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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