Joachim W. Deitmer

Motor control in three types of ciliary organelles in the ciliateStylonychia

Summary1.The motor activity of three types of ciliary organelles (compound cilia), membranelles, frontal cirri and marginal cirri, of the hypotrich ciliateStylonychia mytilus was analyzed using high-speed cinematography (250 images/s). The cell membrane was voltage-clamped, and step or ramp voltage pulses were applied to study the relationship between membrane polarization and motor performance in the three ciliary organelles simultaneously.2.At zero current (=resting) potential the membranelles beat at a frequency of around 40 to 45 Hz, while frontal and marginal cirri were quiescent. Positive or negative voltage pulses activated the frontal and marginal cirri, but did not significantly alter the beating frequency of membranelles.3.Hyperpolarization of the membrane induced beating of the frontal and marginal cirri with the power stroke directed towards the cell posterior (‘hyperpolarizing ciliary activation’), while depolarization of the membrane induced beating of the frontal and marginal cirri in the reversed direction (‘depolarizing ciliary activation’).4.The threshold for both hyperpolarizing and depolarizing ciliary activation was higher, and the latency was larger, for the frontal cirri than for the marginal cirri. The maximum frequency attained was smaller in the frontal cirri than in the marginal cirri during hyperpolarization (around 25 Hz versus 35 Hz) as well as during depolarization (around 35 Hz vs 45 Hz).5.Voltage ramps from −20 mV to +20 mV with respect to the holding (= resting) potential, rising at rates of 20 mV/s to 120 mV/s caused very small — if any — transient changes in the beating frequency of the membranelles. Responses to voltage ramps exhibited similar frequency-membrane potential relationships as step pulses: both frontal and marginal cirri had much the same frequency-voltage pattern, although different absolute beating frequencies (see above).6.The performance of the three types of ciliary organelles is discussed in relation to membrane voltage and current. It is concluded that the membranelles have an unidentified mechanism of motor control, while the activity of both frontal and marginal cirri is coupled to the membrane potential, as also suggested inParamecium.

Concentration-dependent effects of Ba2+ on action potential and membrane currents in the ciliate Stylonychia

Abstract 1. 1. Action potentials and ionic membrane currents of the hypotrich ciliate Stylonychia mytilus were investigated in solutions of various Ba 2+ and Ca 2+ content, keeping the total concentrations of divalent cations constant. 2. 2. All ionic currents-early inward, delayed outward and the current upon membrane hyperpolarization-appeared to be affected by Ba 2+ . 3. 3. Both the inward and the outward steady-state rectification of the membrane were considerably reduced by Ba 2+ . 4. 4. Due to a reduced K + outward current and possibly a decreased inward current inactivation an increasingly large net inward current became apparent in Ba 2+ solutions. 5. 5. The maximum of the early inward current-voltage relationship was reduced and shifted along the voltage-axis in dependence of the Ba 2+ /Ca 2+ concentration ratio. 6. 6. The changes in the membrane currents following gradual substitution of Ca 2+ by Ba 2+ appear to be responsible for the observed increase in both the amplitude and the duration of the action potentials. 7. 7. Our results suggest that the membrane currents are altered due to differences in the interaction of Ca 2+ and Ba 2+ with ionic channels.

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics.

Voltage and time characteristics of the potassium mechanoreceptor current in the ciliateStylonychia

Summary1.The membrane current underlying the hyperpolarizing receptor response following a mechanical stimulus to the cell posterior of the hypotriche ciliateStylonychia mytilus was investigated with a two microelectrode voltage-clamp technique.2.The relationship between the amplitude and the time course of the receptor current and the wave form of the voltage pulse driving the mechanical stimulation system was studied (Figs. 2, 3, 4). The amplitude of the receptor current increased approximately linearly with the driving pulse, but both amplitude and time course of the receptor current were unaffected by varying the duration of the driving pulse.3.The mean maximum amplitude of the receptor current elicited by mechanical stimuli was 20.4 ± 5.6 nA (± S.D., n=19) at the normal resting potential of −51.4±1.6 mV (n=20). This corresponds to an averagemaximum conductance increase of 0.61 μS. The receptor current flow reversed its direction at a membrane potential of −87.7±3.3 mV (n=18; Figs. 5, 6). At the extracellular K+ concentration of 1 mM, the intracellular K+ concentration was calculated to be 33.3 mM.4.The amplitude of the receptor current changed linearly with the membrane potential in the hyperpolarizing direction. In the depolarizing direction the receptor current amplitude increased less than expected from the increased driving force. The conductance increase following a mechanical stimulus was in average less than 50% at zero mV as compared with that at the normal resting potential of around −50 mV.5.The receptor current decayed with a single exponential time course, its mean time constant was 7.3±1.2 ms (n=15). The time course of the receptor current changed with the membrane voltage. The rise time and the time constant of decay of the receptor current increased with hyperpolarization towards the reversal potential, and both decreased with depolarization (Figs. 7, 8). These changes with membrane potential were approximately exponential; the voltage displacement to achieve an e-fold increase of the rise time was −410 mV, and of the decay time constant −110 mV. At membrane potentials just beyond the reversal potential both rise time and decay time constant of the receptor current, now flowinginward, were reduced by 20 to 50%, before increasing again with further hyperpolarization (Fig. 9).6.The results indicate that the life time and possibly the conductance of mechanically activated ionic channels are dependent on the membrane voltage. The implications of these findings for a mechanoreceptor are compared with data obtained at chemoreceptors, e.g. at neuromuscular junctions and other chemical synapses.

Calcium action potentials in larval muscle fibres of the mothEphestia kühniella Z. (Lepidoptera)

Summary1.The ionic requirements for the production of action potentials in the ventral longitudinal muscle fibres of the flour moth larvaEphestia kühniella Zeller (Lepidoptera) were investigated.2.The amplitude and maximal rate of rise of the action potential evoked by indirect stimulation declined when the extracellular Ca-concentration was reduced (Fig. 1).3.Action potentials elicited by intracellularly applied current pulses could be generated in the absence of extracellular Na and Mg.4.Neither TTX (1.5×10−5 g/ml; Fig. 2) nor procaine (15 mM) blocked the action potentials.5.The generation of action potentials could be prevented by omitting all extracellular Ca (Fig. 3).6.The action potential overshoot varied with [Ca++]o, having a slope of 24 to 28 mV for a tenfold change in [Ca++]o (Fig. 5).7.La (1 mM) irreversibly blocked the action potentials (Fig. 7).8.Both Ba and Sr could replace extracellular Ca in the generation of action potentials (Fig. 8).

Effects of cobalt and manganese on the calcium-action potentials in larval insect muscle fibres (Ephestia kühniella)☆

Abstract The effects of cobalt and manganese ions on the calcium action potentials in larval insect muscle fibres were investigated. 1. 1. In either cobalt or manganese (5–25 mM) containing solutions, action potentials could still be elicited by direct stimulation. The maximal rates of rise and fall of these action potentials were however considerably reduced. 2. 2. The repolarization phase of the action potentials was delayed by cobalt and manganese. 3. 3. The membrane input resistance increased in the presence of cobalt and manganese (25 mM) by a factor of 2–4, while the membrane rectification was abolished 4. 4. It is suggested that cobalt and manganese ions reduced both the inward and the outward current of the muscle membrane. These effects of cobalt and manganese are compared with those described for other excitable cells.

Inhibition of a voltage-dependent Ca current by concanavalin A

SummaryIncubation of the hypotrichous ciliateStylonychia mytilus in fluorescein-labeled concanavalin A (Con A, 0.1–0.5 μg/ml) produced a strong fluorescence of its membranelles, but comparatively weak fluorescence of the other compound cilia and of the somatic membrane. Compared to untreated cells, the frequency of spontaneous backward movements was reduced in the presence of 0.5 μg/ml ConA. In electrophysiological experiments Con A altered the excitability of the cell membrane. The two-peak action potential lost its second component which is associated with voltage-dependent Ca channels in the membranelles. The corresponding Ca current (Ca current I) was inhibited by low concentrations of Con A (0.2–0.5 μg/ml). A second voltage-dependent Ca current (Ca current II) was not affected. Reducing the K outward current by intracellular Cs and/or extracellular tetraethylammonium, or changing the holding potential, did not restore the Con A-sensitive Ca current I. Con A also inhibited this current when Ca was replaced by Ba. The inhibitory effect of Con A on the voltagedependent Ca current I was prevented by 10–30 mM α-methyl-d-mannoside, and the lectin wheat germ agglutinin (20 μg/ml) did not affect the Ca currents, indicating that the Con A effect was mediated by binding to specific sugar residues on the excitable membrane. The succinylated dimeric derivative of Con A did not inhibit Ca current I up to concentrations of 5 μg/ml. It is concluded that the two voltage-dependent Ca currents inStylonychia can be chemically isolated due to their different sensitivity to Con A, which appears to bind preferentially to sites near or at the Ca channel in the membranellar membrane.

Calcium channels of the excitable ciliary membrane fromParamecium: An initial biochemical characterization

SummaryA stopped-flow spectrophotometric technique was used to study the kinetics of Ca flux into ciliary membrane vesicles fromParamecium tetraurelia wild-type and several ‘pawn’ mutants with defective Ca conductances. 15mm Arsenazo III was used as metallochromic indicator and as intravesicular Ca trap. The absolute amount of Ca-permeable vesicles was significantly reduced in preparations from the ‘pawn’ mutants compared to wild-type. However, influx kinetics were identical for vesicles from wild-type and ‘pawn’ mutantParamecia when the fraction of Ca-permeable vesicles was taken into account. Ca influx was rapid with a time constant of about 1.5 sec and an initial saturation rate of arsenazo III of about 50%/single vesicle ×sec−1. Ca influx rates were half-maximal at approximately 20 μm Ca. Comparisons of Ba toxicity tested with a behavioral assay, Ca inward conductances under voltage-clamp conditions and Ca influx kinetics between wild-type and the ‘pawn’ mutants pwA (d4-94), leaky pwB (d4-96) and the double mutant pwA/pwB indicated that Ca transport in all types of ciliary membrane vesicles occurred through similar Ca gates.

Membrane properties of identified embryonic nerve and glial cells of the leech central nervous system

SummaryThe membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was −68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was −83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.

Change in the activation rate of voltage-dependent Ba2+ current by conditioning pre-depolarization

When Ba ions replace Ca ions in the external solution, conditioning depolarizing voltage-clamp pulses slow the activation rate of the fast early inward currents in the ciliateStylonychia, while inactivation is greatly reduced in the presence of Ba.