G. P. Findlay

Pathways for the permeation of Na+ and Cl− into protoplasts derived from the cortex of wheat roots

Sodium permeation into cortex cells of wheat roots was examined under conditions of high external NaCI and low Ca(2+). Two types of K(+) inward rectifier were observed in some cells. The time-dependent K(+) inward rectifier was Ca(2+)-sensitive, increasing in magnitude as external Ca(2+) was decreased from 10 mM to 0.1 mM, but did not show significant permeability to Na(+). However, the spiky inward rectifier showed significant Na+ permeation at Ca(2+) concentrations of 1 and 10 mM. In cells that initially did not show K(+) inward rectifier channels, fast and sometimes slowly activating whole-cell inward currents were induced at membrane potentials negative of zero with high external Na(+) and low Ca(2+) concentrations. With 1 mM Ca(2+) in the external solution, large inward currents were carried by Rb(+), Cs(+), K(+), Li(+), and Na(+). The permeability sequence shows that K(+), Rb(+) and Cs(+) are all more permeant than Na(+), which is about equally as permeant as Li(+). When some K(+) was present with high concentrations of Na(+) the inward currents were larger than with K(+) or Na(+) alone. About 60% of the inward current was reversibly blocked when the external Ca(2+) activity was increased from 0.03 mM to 2.7 mM (half inhibition at 0.31 mM Ca(2+) activity). Changes in the characteristics of the current noise indicated that increased Ca(2+) reduced the apparent single channel amplitude. In outside-out patches inward currents were observed at membrane potentials more positive than the equilibrium potentials for K(+) and Cl(-) when the external Na(+) concentration was high. These channels were difficult to analyse but three analysis methods yielded similar conductances of about 30 pS.

The power of movement in plants: the role of osmotic machines.

The apparent and often spectacular movements of animals and insects, movements of the whole organism in relation to its surroundings arising from internally generated forces, have always been, by their very ubiquity, uppermost in our perception of motion in the living world. Movement in plants, generally of one organ in relation to the whole plant, whilst sometimes spectacular, have often in the past been seen as rather esoteric events, amusing perhaps, but of little importance in the general biological scheme of things. However, this is not so; plant movements are quite widespread in occurrence and all are most probably manifestations of a single physiological process, the change in volume of special motor cells. One particular movement, the opening and closing of stomata, which provides a basic control of photosynthesis, is of fundamental importance to the existence of the whole biosphere.

Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts

An electrogenic pump, a slowly activating K+ inward rectifier and an intermittent, “spiky,” K+ inward rectifier, have been identified in the plasmalemma of whole protoplasts from root cortical cells of wheat (Triticum) by the use of patch clamping techniques. Even with high external concentrations of K+ of 100 m m, the pump can maintain the membrane potential difference (PD) down to −180 mV, more negative than the electrochemical equilibrium potentials of the various ions in the system. The slowly activating K+ inward rectifier, apparent in about 23% of protoplasts, allows inward current flow when the membrane PD becomes more negative than the electrochemical equilibrium potential for K+ by about 50 mV. The current usually consists of two exponentially rising components, the time constant of one about 10 times greater than the other. The longer time constant is voltage dependent, while the smaller time constant shows little voltage dependence. The rectifier deactivates, on return of the PD to less negative levels, with a single exponential time course, whose time constant is strongly voltage dependent. The spiky K+ inward rectifier, present in about 68% of protoplasts, allows intermittent current, of considerable magnitude, through the plasmalemma at PDs usually more negative than about −140 mV. Patch clamp experiments on detached outside-out patches show that a possibly multi-state K+ channel, with maximum conductance greater than 400 pS, may constitute this rectifier. The paper also considers the role of the pump and the K+ inward rectifiers in physiological processes in the cell.

Inward membrane current inChara inflata: II. Effects of pH, Cl−-channel blockers and NH 4 + , and significance for the hyperpolarized state

SummaryThe Cl− component of the voltage- and time-dependent inward current activated by hyperpolarizing the membrane ofChara inflata increases exponentially as the external pH, pHo, is lowered from 7 with the membrane potential difference (PD) kept constant. Lanthanum and anthracene-9-carboxylic acid (A-9-C, a Cl− channel blocker) both blocked the Cl− component and removed the pHo sensitivity of the inward current. Lanthanum, however, also decreased the K+ conductance. The hyperpolarized membrane is depolarized by A-9-C in a manner similar to that caused by the removal of external Cl−. Low external concentrations of NH4+ stimulated the Cl− component of the inward current probably as a result of a change in cytoplasmic pH rather than as a result of a change in cytoplasmic [Cl−], since the effect was observed in Cl−-free solutions. The results show that the membrane PD, at hyperpolarized levels, is most likely determined by two factors: the proton extrusion pump, provided it has a reversal PD more negative than about −300 mV, and a voltage-dependent Cl− leak.

Ion channels in the plasma membrane ofAmaranthus protoplasts: One cation and one anion channel dominate the conductance

SummaryThis report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons ofAmaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier.The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K+) and relative permeability sequence K+>Na+>Cl− (1∶0.16∶0.03); whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of sub-conductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (Vm) is depolarized; at millimolar levels of [Ca2+]cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2mm) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO3−>Cl−>K+>Aspartate (2.04∶1∶0.18 to 0.09∶0.04). The relative permeability for K+ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clampVm at a preferred value in the face of events which would otherwise perturbVm.

Multiple conductances in the large K+ channel from Chara corallina shown by a transient analysis method

The large conductance K+ channel in the tonoplast of Chara corallina has subconductance states (substates). We describe a method that detects substates by monitoring the time derivative of channel current. Substates near to the full conductance tend to have long durations and high probabilities, while those of smaller amplitude occur with less probability and short duration. The substate pattern is similar in cell-attached, inside-out and outside-out patches over a range of temperatures. The pattern changes at high Ca2+ concentration (10 mol m-3) on the cytoplasmic face of inside-out patches. One substate at approximately 50% of the full conductance is characterized by a high frequency of transitions from the full conductance level. This midstate conductance is not a constant proportion of the full conductance but changes as a function of membrane potential difference (p.d.) showing strong inward rectification. We suggest that the channel is a single pore that can change conformation and/or charge profile to give different conductances. The mean durations of the full conductance level and the midstate decrease as the membrane p.d. becomes more negative. Programs for analysis of channel kinetics based on an half-amplitude detection criterion are shown to be unsuitable for analysis of the K+ channel.

Potassium channels in the membrane ofHydrodictyon africanum

SummaryThe potential difference across the membrane ofHydrodictyon africanum was controlled by voltage clamping and positive and negative steps in the PD were applied. For positive steps in the PD to values less negative than a threshold value, there is a PD and time-dependent increase in the outward current which has an S-shaped time course. Following the cessation of these steps, the current reverses instantaneously and declines with a simple time course. These currents show a strong K+ dependence and are blocked by tetraethylammonium (TEA) and nonyltriethylammonium (C9) ions, suggesting that they arise from the opening and then the closing of K+ channels. There is also a PD and time-dependent increase in the inward currents in response to negative steps in the membrane PD. The membrane properties have been described by three current-voltage curves, for the instantaneous current, for the steady-state current and for the current flow when the K+ channels are open. The response of the unclamped or free-running membrane PD to steps of constant current can be accounted for by the observed kinetics of the opening and closing of the K+ channels.

ION CHANNELS IN THE PLASMA MEMBRANE OF PROTOPLASTS FROM THE HALOPHYTIC ANGIOSPERM ZOSTERA MUELLERI

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halophytic angiosperm Zostera muelleri. In whole-cell preparations, both outward and inward rectifying currents that activate in a timeand voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K+. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K+ than to Na+. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K+. In addition, the plasma membrane contains a much larger K+ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl− channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd3+, an effect that is reversible upon washout.

Ion channels in the membrane ofChara inflata

SummaryVoltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H+ efflux pump. Positive steps in PD from the resting level often caused the opening of K+ channels with sigmoid kinetics. The channels began to show opening when the PD≃−120 mV for an external concentration of K+ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K+ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K+. The K+ channels have been identified by the blocking effects of TEA+. Another group of channels, probably Cl− and Ca2+ associated with the action potential open when the PD is stepped to values less negative than ≃−50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl−.

Inward membrane current inChara inflata: I. A voltage- and time-dependent Cl− component

SummaryAn inward current which increases in magnitude over a period of seconds is activated when the membrane ofChara inflata (a green alga) in a K+-conductive state is hyperpolarized by a voltage clamp. The peak current and the half-time of activation are exponentially dependent on membrane potential difference. It was found by using an external Cl− electrode that the component exponentially dependent on potential was due to an efflux of Cl−. The measured current-voltage curves and the kinetics of deactivation of the current showed that other time-dependent components contributed to the net inward current. The “punchthrough” theory of Coster (Biophys. J.5:669–686, 1965) does not adequately explain the inward current since a “punchthrough potential” could not be obtained, and the inward current was distinctly time dependent. The voltage and time dependence of the inward current strongly suggests that the Cl− efflux activated by hyperpolarization is through voltage-gated channels which open more frequently as the membrane is hyperpolarized.