Pharmacology of the Ca<sup>2+</sup>‐dependent K<sup>+</sup> channel in corn protoplasts

Ketchum, Karen A.; Poole, Ronald J.

doi:10.1016/0014-5793(90)81343-m

FEBS Letters

1990

DOI: 10.1016/0014-5793(90)81343-m

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Pharmacology of the Ca²⁺‐dependent K⁺ channel in corn protoplasts

Karen A. Ketchum

Ronald J. Poole

Abstract: We investigated the sensitivity of the Ca2+-dependent K ÷ current, IK(Ca), present in corn protoplasts, to different K + channel blockers. &cca) was inhibited by external Cs + (10 mM), Ba 2+ (10 mM), and quinine (0.5 mM): reagents which block many types of outward-rectifying K+channels. In contrast 4-aminopyridine (5 mM), an inhibitor of delayed rectifier or inactivating K + currents, had no effect. Neither of the peptide toxins, apamin or charybdotoxin, specific for CaZ+-dependent K ÷ channels in animal cells… Show more

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Cited by 17 publications

References 23 publications

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Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

White

Tester

1992

Planta

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Plasma membrane was purified from roots of rye (Secale cereale L. cv. Rheidol) by aqueous-polymer two-phase partitioning and incorporated into planar bilayers of 1-palmitoyl-2-oleoyl phosphatidylethanolamine by stirring with an osmotic gradient. Since plasmamembrane vesicles were predominantly oriented with their cytoplasmic face internal, when fused to the bilayer the cytoplasmic side of channels faced the trans chamber. In asymmetrical (cis:trans) 280∶100 mM KCl, five distinct K(+)-selective channels were detected with mean chord-conductances (between +30 and -30 mV; volyages cis with respect to trans) of 500 pS, 194 pS, 49 pS, 21 pS and 10 pS. The frequencies of incorporation of these K(+) channels into the bilayer were 48, 21, 50, 10 and 9%, in the order given (data from 159 bilayers). Only the 49 pS channel was characterized further in this paper, but the remarkable diversity of K(+) channels found in this preparation is noteworthy and is the subject of further study. In symmetrical KCl solutions, the 49 pS channel exhibited non-ohmic unitary-current/voltage relationships. The chord-conductance (between +30 and-30 mV) of the channel in symmetrical 100 mM KCl was 39 pS. The unitary current was greater at positive voltages than at corresponding negative voltages and showed considerable rectification with increasing positive and negative voltages. This would represent 'inward rectification' in vivo. Gating of the channel was not voltage-dependent and the channel was open for approx. 80% of the time. Presumably this is not the case in vivo, but we are at present uncertain of the in vivo controls of channel gating. The distribution of channel-open times could be approximated by the sum of two negative exponential functions, yielding two open-state time constants (τo, the apparent mean lifetime of the channel-open state) of 1.0 ms and 5.7 s. The distribution of channel-closed times was best approximated by the sum of three negative exponential functions, yielding time constants (τc, the apparent mean lifetime of the channel-closed state) of 1.1 ms, 51 ms and 11 s. This indicates at least a five-state kinetic model for the activity of the channel. The selectivity of the 49 pS channel, determined from both reversal potentials under biionic conditions (100 mM KCl∶100 mM cation chloride) and from conductance measurements in symmetrical 100 mM cation chloride, was Rb(+)≥ K(+)> Cs(+) > Na(+) > Li(+) > tetraethylammonium (TEA(+)). The 49 pS channel was reversibly inhibited by quinine (1 mM) but TEA(+) (10 mM), Ba(2+) (3 mM), Ca(2+) (1 mM), 4-aminopyridine (1 mM) and charybdotoxin (3 μM) were without effect when applied to the extracellular (cis) surface.

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Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

White

Tester

1992

Planta

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Selective block by ?-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

1994

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The efficacy and mechanism of alpha-dendrotoxin (DTX) block of K+ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K+ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K+ (K+o) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K+ current (IK,in), but was ineffective in blocking current through the outward-rectifying K+ channels (IK,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for IK,in, neither altering its activation at voltages negative of -120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of IK,in followed a simple titration function with an apparent K1/2 for block of 2.2 nM in 3 mM K+o. However, DTX block was dependent on the external K+ concentration. Raising K+o from 3 to 30 mM slowed block and resulted in a 60-70% reduction in its efficacy (apparent Ki = 10 mM in 10 nM DTX). The effect of K+ in protecting IK,in was competitive with DTX and specific for permeant cations. A joint analysis of IK,in block with DTX and K+ concentration was consistent with a single class of binding sites with a Kd for DTX of 240 pM. A Kd of 410 microM for extracellular K+ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K+ (K1/2 approximately 1 mM) for IK,in activation; they parallel features of K+ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K+ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K+ inward rectifier.

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Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

1994

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Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K+ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K+ channel blocker TEA+ at a high concentration (20 mM). Barium, another "classical" K+ channel blocker, inhibited K+ currents with a Ki of about 1.3 mM. In contrast to guard cells, the cytosolic Ca2+ level proved to be ineffective in regulating the K+ conductance at hyperpolarization. External Ca2+ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating. A comparison between the K+ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca2+ to control K+ influx in xylem parenchyma cells is interpreted in physiological terms.

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Pharmacology of the Ca²⁺‐dependent K⁺ channel in corn protoplasts

Cited by 17 publications

References 23 publications

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Selective block by ?-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

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Pharmacology of the Ca2+‐dependent K+ channel in corn protoplasts

Cited by 17 publications

References 23 publications

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Selective block by ?-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

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Pharmacology of the Ca²⁺‐dependent K⁺ channel in corn protoplasts