N. A. Walker

Bicarbonate assimilation by fresh-water charophytes and higher plants: I. Membrane transport of bicarbonate ions is not proven

SummaryAlthough it is generally believed thatChara and some fresh-water angiosperms transport bicarbonate ions inwards across their plasma membranes, there has been no direct demonstration of such transport in these plants. The (indirect) arguments for their transporting HCO3− are arguments against the inward diffusion of CO2 at the observed rates. They rest on calculations of the equilibrium concentration of CO2 or of the maximum rate at which CO2 might be produced from HCO3− at the pH of the medium outside the cells. SinceChara acidifies the medium over about half the cell surface during C assimilation, these calculations have been based on questionable premises.We propose a model forChara in which the acidification is attributed to active efflux of H+, and we calculate that both the equilibrium concentration of CO2 and its rate of production outside the cell can be high enough to support the observed rates of C assimilation, without postulating transport of the species HCO3− or H2CO3.Calculations are presented also for alternative models in which there is membrane transport of HCO3−. The first includes symport of H+ with HCO3−, again dependent on active H+ efflux. In the second, there is active electrogenic transport of HCO3−. In this case the low pH in the medium outside the cell is caused by the dissociation of H2CO3 produced by hydration of CO2 which leaks from the cell cytoplasm.All three models are consistent with the observations to date, but the first is more economical of postulates. It can also explain the apparent transport of HCO3− by fresh-water angiosperms such asEgeria.

Amine Uniport at the Plasmalemma of Charophyte Cells" I. Current-Voltage Curves, Saturation Kinetics, and Effects of Unstirred Layers

SummaryIn voltage-clamped cells of the algaeChara andNitella an inward current of positive charge occurs when NH4+ or CH3NH3+ is added to the external medium. There is a simultaneous increase in membrane conductance, agreeing with earlier evidence that the current represents the inward uniport of the amine ion.We have obtained current-voltage curves for this uniport, and show the effect on their shape caused by the unstirred layer of solution adjacent to the cell membrane. The current-voltage curves for CH3NH3+, which are less affected by the unstirred layer, are concave towards the current axis, show no saturation with membrane PD in the range −100 to −300 mV, and show saturation as the concentration is raised. The dependence of current on concentration follows a Michaelis-Menten relation, the parameters having the following values at −200 mV:

Amine uniport at the plasmalemma of charophyte cells II. Ratio of matter to charge transported and permeability of free base

\begin{gathered} V_m = up to 200 mA m^{ - 2} for both substrates \hfill \\ K_M = 3 \mu M for NH_4^ + and 200\mu M for CH_3 NH_3^ + . \hfill \\ \end{gathered}

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells

BothVm andKM depend on membrane potential, approximately as exp(−Fψ/6RT) and exp(Fψ/3RT), respectively.The results suggest a transport channel with a single, selective binding site below the membrane surface and a single potential energy barrier at the center of the membrane.The rate of transport falls as the cell takes up amine, and also varies markedly from culture to culture. The significance of this transport for the biology of the charophyte plant is discussed.

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

SummaryThe electrical properties of theChara cell membrane have been studied using a perfusion method based on that of Williamson, R.E. 1975.J. Cell Sci.17∶655. The vacuole, tonoplast, and inner cytoplasm are removed by a brief rapid perfusion. Electrical properties of the plasmalemma indicate that it remains intact after this perfusion.The membrane potential difference after perfusion and with no ATP was close to the potassium equilibrium potential; the current-voltage characteristic had a slope that was time- and voltage-dependent, indicating that the steady-state potassium conductance increased with depolarization. At −125 mV the membrane conductance of the plasmalemma depended on [K+]0. This dependence was inhibited by perfusing with 2.0mm ATP or by clamping at a more negative membrane potential. The addition of ATP to the perfusion medium of unclamped cells caused a hyperpolarization ofca. 50 mV, presumably by activating the proton pump. In clamped cells, perfusion with ATP caused currents ofca. 20 mA m−2, whose magnitude depended on pH0. ATP induced membrane conductance changes which were variable. 2.0mm ADP inhibited the proton pump. The intersection points of current-voltage characteristics can set limits on the stalling potential; the resulting stoichiometry of the proton pump appears to be 1.5–2.0 H+ per ATP.

Ion permeation in a K+ channel inChara australis: Direct evidence for diffusion limitation of ion flow in a maxi-K channel

SummaryWe have previously reported that inward positive current flows across the plasmalemma of voltage-clampedChara cells when ammonia or methylamine is added to the medium. This is attributed to (inward) uniport of the cation. We have measured the stoichiometric ratio of the quantity of methylamine transported to the quantity of positive charge transported. We find 0.9 mol/faraday from pH 5.7 to 8.5, as expected if the cation flux is much larger than that of free base. The ratio increases progressively above pH 9 as the concentration of free base becomes comparable with that of cation: the fluxes fit those predicted if neutral methylamine has a permeability of 1.8 × 10−5 m/sec. This is comparable with the permeability of the methylammonium ion, 6×10−6 m/sec, at low concentration and −200 mV, as previously reported. Low concentrations of NH4+ are found to inhibit entry of CH3NH3+ when membrane PD is constant. Half maximum inhibition is found at ∼20 μm NH4+, in agreement with the apparentKM for NH4+ binding to its uniporter site. This suggests that NH4+ and CH3NH3+ enter by the same uniporter, competing for binding to its binding site.

Membrane conductance ofChara measured in the acid and basic zones

SummaryCytoplasmic drops, covered by a membrane derived from the tonoplast, were obtained from the internodal cells ofChara australis. Patch-clamp measurements were made on this membrane using the droplet-attached configuration with the membrane patch voltage clamped at values from −250 to 50 mV. Single-channel records, filtered at 5 kHz, were analyzed to elucidate the kinetics of the ion gating reaction of the K+-selective channel. The current-voltage characteristics for single channels exhibit saturation and are shown to be consistent with Läugers theory of diffusion-limited ion flow through pores (P. Läuger,Biochim. Biophys. Acta455:493–509, 1976). The time-averaged behavior of the K+ conductance has a maximum at −100 to −150 mV which is produced by the combination of two distinct mechanisms: (1) The channel spending more time in long-lived closed states at positive voltages and (2) a large decrease in the mean open lifetime at more negative voltages. The channel activity shows bursting behavior with opening and closing rates that are voltage-dependent. The mean open time is the kinetic parameter most sensitive to membrane potential, showing a maximum between −100 to −150 mV. The distribution of open times is dominated by one exponential component (time constant 0.3 to 10 msec). In some cases an additional rapidly decaying exponential component was detectable (time constant=0.1 msec). The closed distributions contained were observed to obtain up to four exponential components with time constants over the range 0.1 to 200 msec. However, the voltage dependence of the closed-time distributions suggests an eight-state model for this channel.

Effects of pH and light on the membrane conductance measured in the acid and basic zones ofChara

SummaryWhole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (Vm) was found to be −59±23 mV (n=23) in 1mm Ko−. The meanVm became more negative as [K−]o decreased, but was more positive than the K+ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltagegated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K+-selective channels. A local minimum in the outward current-voltage curve nearVm=150 mV suggests that these currents are mediated by two populations of K+-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca2+ into the cytoplasm. We identify unitary currents from two K+-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than −175 mV. This current is conducted by poorly selective K+ channels, wherePCl/PK=0.43±0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than −250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials −90