Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Abstract

Summary, The membrane of mechanically prepared vesicles of Chara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1 mM ATP to the bath medium induced a membrane current of about 3.2 mA . m 2 creating a voltage across the tonoplast of about -7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K*-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K + with limiting currents of about + 15 and -30 pA, respectively, as measured in symmetric 250 mM KCI solutions. The channel is virtually impermeable to Na + and CI-. However, addition of Na § decreases the K + currents. The 1-V relationships of the open channel as measured at various K + concentrations with or without Na -added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.

Use of Saccharomyces cerevisiae for patch-clamp analysis of heterologous membrane proteins: characterization of Kat1, an inward-rectifying K+ channel from Arabidopsis thaliana, and comparison with endogeneous yeast channels and carriers.

“…That Na+ might have been acting as a blocking ion, rather than as a poor replacement substrate, as has been described for outward-rectifying K+ channels in fungi, algae, and higher plants (7,17,18), was ruled out by experiments in which Na+, added to the bath in the presence of normal K+ (Fig. 4B), had no effect on the Katl inward current.…”

Depolarization-Activated K+ Channel in Chara Droplets

“…The preparation procedure was described by Bertl (1989) with modifications (Pottosin, 1992). Briefly, both cell ends were cut, and the cell interior was perfused with a solution containing (in m~) :…”

“…On the other hand, there is substantial evidence that the droplet membrane in Chara is at least partially made of tonoplast (Lüthring, 1986;Sakano and Tazawa, 1986;Bertl, 1989). The most frequently detected channel type in tonoplasts of various plant tissues shares some properties with SDR comparable conductance, slow kinetics, and voltage dependence (Hedrich et al, 1986;1988;Hedrich and Neher, 1987).…”

“…Most common in this membrane is the large conductance (about 170 pS in 0.15 M KCI), K+-selective channel, which could be activated by hyperpolarization (cytoplasmic side made negative) and micromolar cytosolic Ca2+ (Lühring, 1986;Laver andWalker, 1987, 1991;Bertl, 1989;Pottosin, 1990Pottosin, , 1992Pottosin et al, 1993). An anion-selective channel of lower conductance was also described in this preparation (Tyerman and Findlay, 1989).…”

Internodal cells of the giant green alga Chara as an expression system for ion channels

“…ld). This was shown by staining of the droplet interior with neutral red after addition of Mg-ATP to the bath, thus inducing ATP-fueled active transport of H ÷ from the cytosolic to the vacuolar side of the tonoplast [22]. Also in some of the reported experiments here, nAChR were activated by superfusion of ostensibly inside-out patches with medium containing the agonist.…”