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Plant and Cell Physiology Advance Access originally published online on October 29, 2007
Plant and Cell Physiology 2007 48(12):1747-1757; doi:10.1093/pcp/pcm147
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Characteristics of Anion Channels in the Tonoplast of the Liverwort Conocephalum conicum

Kazimierz Trebacz1,*, Gerald Schönknecht2, Halina Dziubinska1 and Agnieszka Hanaka1

1 Department of Biophysics, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, PL-20-033 Lublin, Poland
2 Department of Botany, Oklahoma State University, 104 Life Sciences East, Stillwater, OK 74078-3013, USA

*Corresponding author: E-mail, trebacz{at}biotop.umcs.lublin.pl; Fax, +48-81-5375901.


  Abstract

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca2+ activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg2+ and low Ca2+ concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg2+ with K+ in the bath. Sr2+ and Ba2+ were also effective activators of the currents. With a Cl– gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current–voltage characteristics shifted towards the reversal potential of Cl–, indicating Cl– selectivity. Currents almost vanished after substituting Cl– with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl–, Formula and Formula, and much less permeable to malate2–. Anion channels did not respond to ATP addition. cAMP (10 µM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.

Keywords: Anion channel - Conocephalum conicum - Magnesium - Patch-clamp - SV channel - Vacuole

Abbreviations: A9C, anthracene-9-carboxylic acid; AP, action potential; CAM, crassulacean acid metabolism; CDPK, calcium-dependent protein kinase; DIDS, 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid; FV channel, fast vacuolar channel; PKA, protein kinase A; SV channel, slowly activating vacuolar channel; VK channel, vacuolar potassium channel; VCL channel, vacuolar anion channel permeable to chloride; VMAL, vacuolar anion channel permeable to malate.

(Received September 19, 2007; Accepted October 23, 2007)
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