Gating of Cl - Currents in Protoplasts from the Marine Alga Valonia utricularis Depends on the Transmembrane Cl - Gradient and Is Affected by Enzymatic Cell Wall Degradation

Abstract

The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 +/- 5 mV (n = 7) was obtained in symmetrical Cl- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 +/- 5 mV (n = 8). By varying the external and internal Cl- concentration, gating was also shown to depend on the Cl- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl- gradient. The voltage- and Cl--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235-247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.

Xylem ionic relations and salinity tolerance in barley

“…Digital low‐pass Bessel filtering of whole cell currents elicited by positive‐going voltage pulses was performed at a cutoff frequency of 2.9 kHz. Correction of clamped voltages for liquid junction potentials as well as for the voltage drop at the uncompensated series resistance was performed as described before (Binder et al. , 2003); for the sign convention see Wegner and Raschke (1994).…”

Plasma membrane anion channels in higher plants and their putative functions in roots

“…Hyperpolarization‐activated anion channel (HAAC) activity (Fig. 1b) has been recorded in the plasma membrane of Amaranthus tricolor cotyledons (Terry et al ., 1991), mesophyll and epidermal cells of the pea ( Pisum sativum ) leaf (Elzenga & Volkenburgh, 1994, 1997), Chara ( Chara inflata ) (see Tyerman, 1992 for review), the seed coats of Phaseolus vulgaris (Zhang et al ., 2004b), suspension cultured cells from carrot ( Daucus carota ) (Barbara et al ., 1994), A. thaliana (Lew, 1991), barley ( Hordeum vulgare ) (Amtmann et al ., 1997) and Asclepias tuberosa (Schauf & Wilson, 1987), the marine phytoplankton Coccolithus pelagicus (Taylor & Brownlee, 2003) and the marine alga Valonia utricularis (Heidecker et al ., 1999; Binder et al ., 2003). There are few unifying features apparent from these studies, although most currents exhibit voltage‐dependent inward rectification which are mediated by large conductance channels (i.e.…”

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, Ulva pertusa (Chlorophyta)

“…Heidecker et al (1999) isolated protoplasts from Valonia utricularis and demonstrated three Cl − specific channels and at least one specific K + channel. By employing the whole-cell patch-clamp technique, Binder et al (2003) recorded large inward Cl − currents elicited by negativegoing voltage pulses from V. utricularis. By comparing enzymatic treatment with mechanical release, they also showed that enzymatic treatment could affect the gating properties of the ion channel of protoplasts.…”

“…According to the Nernst equation, the calculated reversal potentials for chlorideselective pathways were close to zero because of nearly equal concentrations of Cl − on both sides of the membrane (E Cl =+0.85 mV). This situation is characterized below as "symmetrical 150 mM Cl − " (Binder et al 2003). For ideal potassium selectivity, the calculated reversals, E K , were −61.52 mV.…”

“…Patchclamp research on higher plant species was first reported in 1984 (Schroeder et al 1984). However, the application to marine algae and plants is limited to Valonia utricularis (Heidecker et al 1999;Binder et al 2003), Coccolithus pelagicus (Taylor and Brownlee 2003) and Posidonia oceanica (Carpaneto et al 2004).…”