Ion channel activity during the action potential in Chara: new insights with new techniques.
The dynamics of macroscopic currents underlying the electrically triggered action potential (AP) in the giant alga Chara corallina were directly 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 Ba(2+) to the bath, the excitation current, i.e. the 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 demands 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 several 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 active during an AP. Typically, variable numbers of CI(-) channels activate in a random fashion for short periods of time when favoured by positive voltages in combination with high concentrations of extracellular Ca(2+) (Ca(2+)(o)) 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 short dura- tion of channel activity, as well as the fast rising and somewhat slower trailing kinetics is similar in duration and dynamics to AP-associated changes in membrane permeability of the whole Chara cell to Cl(-) (P(Cl(-))). Taken together, the data stress that the characteristic, transient activation of random numbers of Cl(-) channels seen in membrane patches is the elementary unit of the Cl(-) excitation current. However, due to the random nature of this transient activity, gating of Cl(-) channels can not be explained on the basis of previous models for excitation: gating can neither be due to intrinsic voltage sensitivity of the Cl(-) channels, nor to a voltage-dependent influx of Ca(2+) and subsequent activation of Ca(2+)-sensitive Cl(-) channels. To account for the short life-time and for the randomness of Cl(-) channel activity, the putative gating factors Ca(2+) and voltage must be uncoupled in time. This could be explained by a random release of Ca(2+) from stores, the latter being filled in a voltage-sensitive manner via non-specific cation channels from the outside. A 4 pS non-selective cation channel in the plasma membrane may serve this purpose. The 40 pS K(+) channel, which becomes transiently active in C. corallina during a cell AP, is an outward rectifier. At negative resting voltages the channel has a low open probability (< <1%). At voltages reached during an AP the open probability rises significantly reaching half-maximal open probability at -25 mV. 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(+) (P(K(+))), stressing that the kinetic properties of the 40 pS K(+) channel are responsible for the course of whole cell P(K(+)) changes. Voltage sensitivity of the K(+) channels suggests that they are activated during an AP by the drop in membrane voltage in order to aid repolarization. However, the rise and decay of P(K(+)) during an AP also shares similarity with the time-course of transient changes in cytoplasmic concentration of free Ca(2+), [Ca(2+)](cyt), the latter being measured in parallel experiments with the Ca(2+)-sensitive fluorescent dye, Fura-2, in excited C. corallina cells. This similarity could suggest that gating of the 40 pS K(+) channel is also sensitive to [Ca(2+)](cyt) and that the latter sensitivity is rate-limiting for activity during an AP.
Pflanzenphysiologisches Institut der Universität, Göttingen, Germany.
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Thiel
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Homann
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Plieth