The negative currents observed as the Chara membrane is voltage-clamped to hyperpolarized PD levels. The experimental protocol clamps the PD to resting level for 2 sec, to a hyperpolarized level (shown next to each current trace) for 12 sec and back to the pre-clamp resting level.	Mary Beilby and exchange student Sebastian Westermann.

I was just reading a paper written by my Glasgow colleague, Mike Blatt, who found Ca²⁺ channels in bean guard cells, which are activated by membrane potentials more negative (hyperpolarized) than the usual cell resting potential difference (PD). In charophytes, Ca²⁺ channels are activated by PDs more positive than the resting PD. The inflow of Ca²⁺ into cytoplasmic compartment opens Ca²⁺-activated Cl^- channels, initiating the action potential. The outflow of Cl^- was also observed at hyperpolarized PDs by Hans Coster in 1965. Hans modeled the cell membrane as a pn junction and assumed that the large Cl_^- currents arise from a punchthrough effect, where the depletion layer grows to one edge of the membrane. In 1986 an Adelaide colleague, Steve Tyerman, argued that the time dependence of the large currents at hyperpolarized PDs points to Cl^- channel-mediated conduction and that these channels are activated by negative membrane PDs. Putting all this information together, I asked the obvious questions: are the Ca²⁺ channels in Chara also activated by hyperpolarized PDs and is it the Ca²⁺-activated Cl^- channel that conducts the currents observed? So, this became Sebastian’s project.

The cell PD was controlled by voltage clamp. The rise of calcium concentration in the cytoplasmic compartment was estimated by observing the stoppage of cytoplasmic streaming. The Ca²⁺ channels were blocked by application of LaCl₃. Sebastian mastered all the experimental techniques and approached the project with maturity of a PhD student. He gathered large amounts of excellent data. Yes, the cytoplasmic streaming stopped, while the cells were clamped to PDs more negative than -350 mV. The negative currents increased with greater Ca²⁺ concentration of the medium. The streaming stoppage was abolished by application of LaCl₃, but only some of the negative current. After the negative voltage clamp was released, the cells became more depolarized and the recovery to normal resting PD took many minutes. The amount of depolarization increased as the cell was clamped to increasingly negative PDs. We conclude that the Ca²⁺ channels in Chara are opened by hyperpolarization as well as depolarization and the Cl^- outflow in each case is mediated by Ca²⁺-activated Cl^- channels. However, there is another unexpected effect: the proton pump, which is mainly responsible for the negative resting PD of about -200 mV, is inhibited by the voltage clamps to negative levels.

Mary Beilby