Properties of Single K⁺ and Cl⁻ Channels in Asclepias tuberosa Protoplasts

Abstract

Potassium and chloride channels were characterized in Asckpias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole (2,3,11,13,26,30), and there is evidence of Ca2"-dependent second messengers (1,5,14,(18)(19)(20). Plant hormones also affect nonspecific membrane permeability and ion channels (23,24,29).Until recently cell walls hindered direct biophysical studies of ion channels in plants, but patch-clamp of enzymically isolated protoplasts has made it possible to resolve single ion channels (16,27). Viciafaba guard cells were shown to have K+ channels with a conductance of 20 to 30 pS2 (27), large enough to produce net K+ fluxes sufficient to account for ABA-dependent stomatal closure (15,28). In a related study, K+-selective cyclic polyethers inhibited stomatal opening, presumably by short-circuiting K+ channels normally present (12). Four distinct channels were observed in wheat protoplasts including a 35 to 40 pS voltage-' Supported by National Science Foundation Grant BNS 86-96095, the Whitehall Foundation, the Indiana Academy of Science, and the Project Development Program of Indiana University.Abbreviations: pS, picosiemens; TEA', tetraethylammonium; pA, picoamperes. dependent channel and a larger 160 to 180 pS channel (16), the latter having a conductance similar to Ca2"-activated K+ channels in animal cells. Channels carrying maleate and K+ were seen in vacuoles from barley leaf mesophyll protoplasts (9), while TEA'-sensitive and TEA'-insensitive K+ channels with singlechannel conductances of 100 and 30 pS, respectively, have been observed in protoplasmic droplets from Chara corallina (1 1).We used whole-cell and excised-patch voltage-clamp to demonstrate voltage-dependent TEA' and Cs' sensitive K+ channels, as well as Zn2+ and ABA-sensitive Cl-channels in protoplasts derived from Asclepias tuberosa suspension cell cultures. The presence of hormone-responsive ion channels in an experimental situation that permits control of internal and external composition should prove ideal for studying the role of ion channels in plant physiology and development. MATERIALS AND METHODSCell suspension cultures were derived from callus ofAsclepias tuberosa by methods previously described (4,21). Suspension cultures were maintained on a rotary shaker in liquid MurashigeSkoog medium (17), supplemented with 5.0 mg/L adenine, 0.5 mg/L 2,4-D, 0.5 mg/L benzyl adenine, 0.1 gm/L myoinositol, 0.4 gm/L casein hydrolysate, and 2% sucrose (pH 5.8, see Ref. 4). Cultures were maintained under fluorescent ceiling lights at 25°C by transferring 5 ml aliquots to 20 ml fresh medium every 1 to 2 weeks. Rapidly growing cultures were obtained by transferring cells to fresh medium more often. To obtain protoplasts, 6 to 14 d old cultures were filtered through 400 4m sterile gauze, centrifuged at 5000 rpm for 10 min, and the pellet resuspended and digested for 4 to 12 h at 25°C (on a reciprocal shaker) in an equivolume (25 ml) solution of 0.5% Macerase and 1.0% Cellulysin (Calbiochem) dissolved in 350 mM mannit...

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.…”

Localized Patch Clamping of Plasma Membrane of a Polarized Plant Cell

“…Careful control of cell turgor enabled a small portion of plasma membranebound cytoplasm to be exposed. (1,11,17,19,21), and more recently, the technique has been used to investigate their regulation (7,10,15,16,18,20).…”

“…The purity of the enzymes is crucial to avoid degradation of the plasma membrane lipids and proteins. Reported success rates for obtaining high resistance (>5 GO) seals on enzymically isolated protoplasts vary but are generally low (5,19). Attempts to improve the low success rates have often proved unsuccessful (6), although recent improvements to the procedure of enzymically isolating protoplasts may increase the number of successful GU seals (4).…”