Bernhard U. Keller

Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells

In the molecular layer of the cerebellar cortex, Purkinje cells and interneurons receive a common excitatory input from parallel fibers. The AMPA/kainate receptor-mediated parallel fiber excitatory postsynaptic current (EPSC) recorded in Purkinje cells decays much more slowly than that recorded in interneurons. We show that this slowness of decay does not result from dendritic filtering and that it is unlikely to reflect the deactivation kinetics of the postsynaptic receptors. Agents blocking glutamate uptake prolong the EPSC in Purkinje cells. We conclude that the slow EPSC decay results from the continued presence of transmitter glutamate. This may be due to retarded transmitter diffusion around spines or to cross-talk between neighboring active synapses.

A membrane fusion strategy for single-channel recordings of membranes usually non-accessible to patch-clamp pipette electrodes

Membranes of cellular organelles and plasma membranes of some type of cells are not accessible to the high‐resolution recordings that the conventional patch‐clamp technique allows. However, when these purified membranes are dehydrated together with small lipid vesicles and hydrated again, cell‐size vesicles (5–100 μm diameter) are obtained, on which single‐channel recordings are possible. This approach, which has been proven successful with about ten different membrane preparations of varied origin, is further illustrated with two examples. First, a known conductivity of the sarcoplasmic reticulum membrane is compared with data obtained by using other techniques. Second, a new sodium current, present at purified postsynaptic membranes from the Torpedo electric organ, is described.

Patch clamp analysis of excitatory synapses in mammalian spinal cord slices.

Excitatory synaptic transmission to visually identified α-moto neurones was studied in thin slice preparations of the neonatal rat spinal cord. Excitatory postsynaptic currents (EPSCs) elicited by stimulation of intraspinal presynaptic fibres were recorded using the whole-cell patch clamp technique, following blockade of inhibitory transmission by bath application of strychnine and bicuculline. The EPSCs could be separated pharmacologically into N-methyl-d-aspartate- (NMDA) and non-NMDA-receptor-mediated components, where the contribution of the NMDA-mediated component was significant only at holding potentials more positive than −50 mV. Graded stimulation of intraspinal fibres showed that the NMDA- and the non-NMDA-mediated EPSCs were evoked by activation of presynaptic fibres with similar sensitivities to the stimulation intensity, suggesting that the same presynaptic fibres released the excitatory amino-acid (EAA) activating the two sub-sets of receptors. Studies of the amplitude fluctuations of EPSCs elicited by stimulation of a presumed single fibre revealed similar proportions of transmission failures and similar distributions of both the NMDA- and the non-NMDA-mediated components. These similarities suggest that the EAA transmitter activating the two sub-types of receptors is released from the same set of synaptic boutons and that the receptors are therefore post-synaptically co-localized. In addition the gamma aminobutyric acidB (GABAB) receptor agonist l-baclofen, which is known to decrease transmitter release, changed the amplitude distributions of non-NMDA- and NMDA-receptor-mediated EPSCs into unimodal distributions without affecting the amplitude of the presumed unitary event. The similarity between the transmitter release profiles of the two EAA components further supports the notion of postsynaptic receptor co-localization.

Single channel recordings of reconstituted ion channel proteins: an improved technique

Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This “hydration technique” consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K+-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K+-channel studied displays a conductance γK+ of 145 pS. The single channel conductance in 100 mM Rb+ and Na+ is γRb+ = 98 pS and γNa+ = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K+-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed.

Further investigation on the high-conductance ion channel of the inner membrane of mitochondria

By use of the patch-clamp technique, the inner membrane of mouse liver and heart mitochondria is shown to contain a highly conductive (around 100 pS in symmetrical 150 mM KCl) and voltage-dependent ion channel. This channel closely resembles that previously found in cuprizone-treated mouse liver inner mitochondrial membrane. The paper discusses the electrical properties of the channel and its possible physiological function. The reconstitution in giant liposomes of a partially purified ox heart inner membrane fraction containing the channel and the use of various inhibitors are also presented.

Patch clamp techniques to study ion channels from organelles.

Publisher Summary This chapter describes several strategies to isolate intracellular membranes and yield organelles large enough for patch clamp experiments, and focuses on the methods utilized for patch clamping membranes of mitochondria, endoplasmic reticulum, and plant vacuoles. The chapter also discusses the potential application of these techniques for other intracellular membranes. Three principally different strategies to patch clamp intracellular ion channels are proposed in this chapter. One method, which is preferably used for highly folded membranes, is the osmotic swelling technique, and it has been successfully applied to membranes from mitochondria and thylakoids. The dehydration/rehydration cycle composing the hydration technique provides a general approach to investigate intracellular ion channels, and provides a basis to investigate ion channel structure and function in its original environment. In some cases, when the organelles are large enough, they can be patch damped directly without any manipulation to enlarge their size. In the case of plant vacuoles mechanically isolated from plant tissue, they can be patch clamped in an osmotically adapted bathing solution. Taken together, these experiments demonstrate that there is no general strategy for patch damping different organdies.

Voltage-Dependent Channels are Present in the Inner Membrane of Mitochondria

The study of mitochondria with electrophysiological methods has been generally hampered by the small size of normal mitochondria and by the lack of suitable techniques applicable to such minute organelles. An attempt of this kind was nonetheless made by Tedeschi and coworkers (Tedeschi, 1980), who introduced microelectrodes into enlarged mitochondria, obtained from the liver of mice fed with the copper chelator agent oxalic acid (ciclohexylidenehydrazide), known as cuprizone. Contrary to some expectations (Ferguson and Sorgato, 1982, and references therein), a respiration dependent membrane potential was not detected with this technique (Campo et al., 1984). One possible reason for such result could be inherent to the impalement method itself, i.e. in the recognised difficulty of obtaining tight seals between the electrode and the membrane. Ions would then be free to permeate through the leak conductance and thus collapse the membrane potential. Recently, the patch-clamp technique has become available (Neher and Sakmann, 1976). This method is less invasive than the inpalement of microelectrodes and the relative ease of seal formation in the gigaohm resistance range makes the recording of low intensity currents feaseable (Hamill et al., 1981). With this in mind, we have tried to patch-clamp mitochondria deprived of the outer membrane. Our attempt was successful as we were able to record elementary currents through single channels from the inner membrane. It was found that these channels are voltage-dependent and slightly more permeable to chloride than to potassium ions. Interestingly, similar electrical properties were displayed by an as yet unidentified protein which coprecipitates with the membrane integrated part Fo, the membrane-associated and proton conductive part of the ATP syntase complex (Amzel and Pedersen, 1983) during the isolation procedure of the enzyme.

Molecular Mechanisms of Ion Transport: New Insights by Patch-Clamp Studies

New insights into the molecular processes involved in ion and nutrient transport across membranes of animal and plant cells were obtained since the application of the patch-clamp technique to isolated cells, protoplasts (wall-free plant cells) and organelles. While excitable electrical behaviour was first observed in plant cells about a century ago, the underlying mechanisms are only now being directly studied at the molecular level. Ion channels are integral transmembrane proteins which, when open, allow the movement of ions and some non-electrolytes down their electrochemical potential gradients. Although ion currents in plant cells were among the first to be studied in detail, the electrophysiological characterization of plant ion channels has been somewhat slower compared to their animal counterparts. This has been due to problems specific to plants, such as the presence of the cell wall, having the plasma membrane and vacuolar membrane in series separated by only a relatively small cytoplasmic compartment.