ION-CHANNEL ACTIVITY DURING THE ACTION-POTENTIAL IN CHARA - NEW INSIGHTS WITH NEW TECHNIQUES

The dynamics of macroscopic currents underlying the electrically trigg ered action potential (AP) in the giant alga Chara corallina were dire ctly recorded with an action potential clamp method. In this technique an AP is recorded and repetitively replayed as the command voltage to the same cell under voltage control. Upon adding the channel blockers niflumic acid and/or Ba2+ to the bath, the excitation current, i.e. t he current crossing the membrane during an AP, can be dissected into a transient, fast-appearing Cl- inward current and a transient delayed K+ outward current. The delayed onset of the K+ outward current demand s the postulation of an additional outward current in order to balance the excess Cl- inward current at the onset of the AP. The capacitive current that alters the charge on the membrane during excitation is se veral orders of magnitude too small to be relevant for charge balance. Measurements of single channel activity in the plasma membrane of C. corallina by the patch clamp method shows two types of Cl- channel (15 and 38 pS with 100 mM Cl- in the pipette) and one type of K+ channel (about 40 pS with 100 mM K+ in the pipette) which become transiently a ctive during an AP. Typically, variable numbers of Cl- channels activa te in a random fashion for short periods of time when favoured by posi tive voltages in combination with high concentrations of extracellular Ca2+ (Ca-o(2+)) or during an AP of the whole cell. The peak values of these Cl- channel currents measured in a patch are such that they can account quantitatively for the peak of the whole cell Cl- excitation current studied under comparable ionic conditions. Furthermore, the sh ort duration of channel activity, as well as the fast rising and somew hat slower trailing kinetics is similar in duration and dynamics to AP -associated changes in membrane permeability of the whole Chara cell t o Cl- (PCl-). Taken together, the data stress that the characteristic, transient activation of random numbers of Cl- channels seen in membra ne patches is the elementary unit of the Cl- excitation current. Howev er, due to the random nature of this transient activity, gating of Cl- channels can not be explained on the basis of previous models for exc itation: gating can neither be due to intrinsic voltage sensitivity of the Cl- channels, nor to a voltage-dependent influx of Ca2+ and subse quent activation of Ca2+-sensitive Cl- channels. to account for the sh ort life-time and for the randomness of Cl- channel activity, the puta tive gating factors Ca2+ and voltage must be uncoupled in time. This c ould be explained by a random release of Ca2+ from stores, the latter being filled in a voltage-sensitive manner via non-specific cation cha nnels from the outside. A 4 pS non-selective cation channel in the pla sma membrane may serve this purpose. The 40 pS K+ channel, which becom es transiently active in C. corallina during a cell AP, is an outward rectifier. At negative resting voltages the channel has a low open pro bability (< < 1%). At voltages reached during an AP the open probabili ty rises significantly reaching half-maximal open probability at -25 m V. The elevated activity of the 40 pS channel associated with membrane excitation relaxes at the end of an AP with a time constant of about 2.5 s. A comparable time constant of 2 s can be obtained for the decay of the transiently elevated permeability of the membrane to K+ (PK+), stressing that the kinetic properties of the 40 pS K+ channel are res ponsible for the course of whole cell PK+ changes. Voltage sensitivity of the K+ channels suggests that they are activated during an AP by t he drop in membrane voltage in order to aid repolarization. However, t he rise and decay of PK+ during an AP also shares similarity with the timecourse of transient changes in cytoplasmic concentration of free C a2+, [Ca2+](cyt), the latter being measured in parallel experiments wi th the Ca2+-sensitive fluorescent dye, Fura-P, in excited C. corallina cells. This similarity could suggest that gating of the 40 pS K+ chan nel is also sensitive to [Ca2+](cyt) and that the latter sensitivity i s rate-limiting for activity during an AP.