Potassium Channels in Motor Cells of Samanea saman

Abstract

ABSTRACILeaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K+, Cl and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K currents that flow during leaflet movement. Recordings in the "whole-cell" mode reveal depolarization-activated K+ currents in extensor and flexor cells that increase slowly (t½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K+ currents is adequate to account quantitatively for K+ loss, previously measured in vivo during cell shrinkage. The K+ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light-and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.Leaflet movements in nyctinastic (night closure) plants often involve significant changes in the volume and up to severalfold variation in the ionic content of motor cells in the pulvinus (reviewed in Ref. 21). These variations may occur in response to light, darkness, and an endogenous biological clock. Cells in the extensor region of the pulvinus take up K+ and Cl-as they swell during leaflet opening, and lose both ions as they shrink during leaflet closure, while cells in the opposing (flexor) region behave in the reverse manner (12,22,23,25,30,32).We undertook this study to examine a possible role for K+ channels in leaflet movement-related K+ fluxes and changes in cell volume in the nyctinastic legume Samanea saman. K+ channels have already been described in giant algae (1, 5) and in protoplasts isolated from wheat mesophyll cells (13,14), Vicia faba guard cells (27,29) Protoplast Isolation. Terminal secondary pulvini from the fourth to ninth leaf (counting from the apex) were harvested 2 to 3 h after the beginning of the light period in the growth chamber, or 2 to 3 h after sunrise in the greenhouse. Protoplasts were prepared by enzymic digestion of slices of extensor or flexor tissue (pooled separately), as described in (7) but with the following modifications. (a) The osmotic pressure of the pre-digestion solution was raised in two steps to 680 mosmol to ensure plasmolysis. (b) Pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan) was added to the digestion solution (which contained cellulase, pectinase and Driselase) to a final concentration of 0.2% (w/v). (c) A second purification step was added, as follows: the cells collected from the Ficoll interface were layered again on a sucrose gradient (2), spun at 60 to IOOg for 5 min, and collected and kept on ice for up to 24 h for patch-clamp experiments.Forty to fifty protoplasts of each type, flexor and ...

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

“…The patch-clamp technique is described in detail by Hamill, Marty, Neher, Sakmann, and Sigworth (1981). The application to plant cell protoplasts is described in detail by Moran et al (1988), and by Moran, Fox, and Satter (1990). Briefly, a drop of the protoplast suspension was added to ~ 200 p.1 of the recording solution, the protoplasts were allowed to settle and stick to the glass bottom of the recording chamber and then washed with ~ 2-3 ml of the recording solution.…”

Roles of Higher Plant K+ Channels

“…A depolarization-activated K+ channel in protoplasts derived from motor cells is capable of carrying the large K+ currents that flow from shrinking cells, is sensitive to inhibition by TEA and quinine, and conducts severa1 monovalent cations with a selectivity sequence K+ > Rb+ > Na' -Cs+ -Li+ (Moran et al, 1988). As explained above, to maintain a significant K+ efflux the membrane voltage must be more positive than E, .…”

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

“…The Ca 2 +-dependent K ÷ channel found in corn suspension cells is inhibited by all 4 reagents ( [6], and data presented). These results sup- [4] saman [5] muscipula [3] corallina [19] (+) Inhibition; (-) no effect; In most cases, IC50 has not been determined. The concs, given are those tested.…”

“…One current which is prominent in many protoplast preparations is the voltage-and time-dependent K ÷ current (IK+out) elicited by membrane depolarization [1]. Although IK+out recorded in different types of cells exhibits similar activation kinetics and has a comparable activation range [2][3][4][5][6][7], the degree of homology between the K ÷ channel proteins in protoplasts derived from different species and/or tissues is unclear. The range in unitary conductance, 3 to 65 pS [3][4][5][8][9][10] recorded for plant plasmalemma K ÷ channels, and the observation that multiple regulatory mechanisms (e.g.…”

“…Although IK+out recorded in different types of cells exhibits similar activation kinetics and has a comparable activation range [2][3][4][5][6][7], the degree of homology between the K ÷ channel proteins in protoplasts derived from different species and/or tissues is unclear. The range in unitary conductance, 3 to 65 pS [3][4][5][8][9][10] recorded for plant plasmalemma K ÷ channels, and the observation that multiple regulatory mechanisms (e.g. cytosolic Ca 2÷ [10,11] or abscisic acid [8,12]) can modulate K ÷ currents in some protoplasts, suggest that more than one type of K ÷ channel may be responsible for IK+out in these diverse cell systems.…”