A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis

Abstract

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant "mother cells" (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123-134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl(-) channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K(+) channel (VKC1, Valonia K(+) Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl(-) media, VAC1 was most frequently observed and had a single channel conductance of 36 +/- 7 pS (n = 4) in the outside-out and 33 +/- 5 pS (n = 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 +/- 4 mV (n = 4, outside-out) and 9 +/- 7 mV (n = 10, cell-attached) close to the Nernst potential of Cl(-) and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl(-) media (bath/pipette; E(Cl(-)) -20 +/- 7 mV (n = 4); Nernst potential -28 mV). Consistent with a selectivity for Cl(-), VAC1 was inhibited by 100 micronM DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl(-) conditions yielded a midpoint potential of -12 +/- 5 mV (n = 4, outside-out) and -3 +/- 6 mV (n = 9, cell-attached) and corresponding apparent minimum gating charges of 15 +/- 3 (n = 4) and 18 +/- 5 (n = 9). The midpoint potential shifted to more negative values in the presence of a Cl(-) gradient. VAC2 was activated by voltages more negative than E(Cl(-)) and was always observed together with VAC1, but less frequently. It showed a "flickering" gating. The single channel conductance was 99 +/- 10 pS (n = 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 +/- 5 pS (n = 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 +/- 27 pS (n = 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl(-) conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.

Touch Sensing and Thigmotropism

“…Apart from this common mechanism of gating, however, the observed channel activities are quite diverse with regard to selectivity, conductance, or voltage dependence. Some channels are unselective (Spalding and Goldsmith 1993) and others have been demonstrated to show preference for anions (Falke et al 1988;Cosgrove and Hedrich 1991;Heidecker et al 1999;Qi et al 2004) or cations (Garrill et al 1994), whereas certain channels show high selectivity for K + or Ca 2+ (Cosgrove and Hedrich 1991;Ding and Pickard 1993;Liu and Luan 1998;Dutta and Robinson 2004). Measured conductances range from 3 to 15 pS for Ca 2+ -selective channels (Cosgrove and Hedrich 1991;Ding and Pickard 1993;Dutta and Robinson 2004) to 13 to 97 pS for anion channels (Falke et al 1988;Cosgrove and Hedrich 1991).…”

“…Measured conductances range from 3 to 15 pS for Ca 2+ -selective channels (Cosgrove and Hedrich 1991;Ding and Pickard 1993;Dutta and Robinson 2004) to 13 to 97 pS for anion channels (Falke et al 1988;Cosgrove and Hedrich 1991). Although most channels are seemingly independent of membrane potential (Falke et al 1988;Garrill et al 1994;Liu and Luan 1998;Heidecker et al 1999;Dutta and Robinson 2004;Qi et al 2004), others show significantly reduced open probabilities at either negative or positive voltages (Cosgrove and Hedrich 1991;Spalding and Goldsmith 1993), whereas some are active only at higher voltages, irrespective of the polarity (Ding and Pickard 1993).…”

Electrophysiology of Turgor Regulation in Marine Siphonous Green Algae

“…It also suggests that Cl ) crosses the membrane by different mechanisms under hypo-and hypertonic stress conditions. Patch clamp experiments (Heidecker et al, 1999) provide evidence that DIDS is a Cl ) channel inhibitor in this organism, although the relationship between the channel measured in protoplasts and activities in intact, turgid cells is unclear. Since protoplasts have no turgor, they would be expected to be responding as though to hypertonic stress, if turgor or pressure is the signal that initiates turgor regulation.…”

“…The electrical responses seen at the tonoplast could be the response to turgor change, and not to its initial detection. Patchclamp experiments (Heidecker et al, 1999) identified a stretch-activated channel in the plasma membrane, and this could well play the role of a turgor sensor (Bisson & Gutknecht, 1980;Pickard & Ding, 1992). …”

“…The mechanism by which a cell measures its turgor is not known and numerous hypotheses have been proposed (Coster & Zimmermann, 1976;Gutknecht et al, 1978;Pickard & Ding, 1993;Bisson & Kirst, 1995;Heidecker et al, 1999;Shepherd et al, 2002;Kacperska, 2004). These hypotheses generally invoke physical differences that occur as a result of pressure on or differential pressure across the membrane, such as proximity between elements of the membrane and cell wall, curvature or compression of the membrane, and tension within the membrane.…”