AN ANION CURRENT AT THE PLASMA-MEMBRANE OF TOBACCO PROTOPLASTS SHOWS ATP-DEPENDENT VOLTAGE REGULATION AND IS MODULATED BY AUXIN

Plasma membrane ion channels of protoplasts from tobacco cell suspensions were characterized by patch-clamp experiments. In the whole-cell configuration, a voltage-dependent current with a current maximum around -90 mV was observed that displayed a reversal potential close to the Nernst potential for chloride. This whole-cell current was identified as an anion current by replacing the internal Cl- with glutamate. The tobacco suspension anion channel (TSAC) was characterized by fast activation/deactivation and slow inactivation kinetics with voltage-dependent time constants in the range of milliseconds and seconds, respectively. Among the plant channels, TSAC exhibits original properties in terms of phosphorylation-dependent voltage regulation while sharing similarities with the fast anion channel from stomatal guard cells (GCAC1). The voltage dependence of the whole-cell current reflecting the fast deactivation of the current at potentials of less than -100 mV was observed only in the presence of internal ATP or when ATP was replaced by the protein phosphatase inhibitor okadaic acid, and was suppressed by staurosporine. This suggests that protein phosphorylation may be involved in regulating the activity of the anion channel. As observed on GCAC1 the active auxin 1-NAA caused a time- and concentration-dependent shift of the activation potential of TSAC. In addition, TSAC reacted to the auxin agonist antibody D16 providing evidence for the recognition of the auxin signal at the outer face of the plasma membrane.