A simple technique for transferring excised patches of membrane to different solutions for single channel measurements

Quartararo, Nino; Barry, Peter H.

doi:10.1007/bf00581332

Cited by 11 publications

(4 citation statements)

References 11 publications

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“…For the whole-cell recordings, drugs were delivered to individual cells from a "puffer" pipette with a tip opening of about 10 pm, driven by a Picospritzer (General Valve Corp., Fairfield, NJ). In the excised-patch experiments, the electrode with the patch at its tip was moved through the air from the culture dish to an isolated recording chamber by means of the "sleeve" technique of Quartararo and Barry (1987). A polythene sleeve placed on the pipette shaft could be slipped down over the tip, trapping a drop of solution by surface tension and thus keeping the membrane patch covered with saline solution during transfer.…”

Section: Methodsmentioning

confidence: 99%

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta

Zufall¹,

Stengl²,

Franke³

et al. 1991

J. Neurosci.

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Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 +/- 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 microM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (gamma = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP greater than cGMP greater than cAMP, with an inhibition constant for ATP of Ki = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 microM induced the opening of a Ca2(+)-activated K+ channel (gamma = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (gamma = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.

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Section: Methodsmentioning

confidence: 99%

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta

Zufall¹,

Stengl²,

Franke³

et al. 1991

J. Neurosci.

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“…Methods for achieving these membrane patch configurations are described by Hamill et al (1981). Exchange of bathing media was achieved using the "sleeve" technique of Quartararo and Barry (1987) whereby the excised patch is transferred between baths containing different solutions. During experiments on drop-attached patches the composition of the medium bathing the cytoplasmic drops was 150 mol/m 3 KC1, 5 mol/m 3 CaCI2, 5 mol/m 3 H+MES, adjusted to pH 5.2 (the vacuolar pH) using KOH.…”

Section: Methodsmentioning

confidence: 99%

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

1989

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We report a study of a potassium-selective channel in the membrane delineating cytoplasmic drops from Chara australis. The relatively large conductance (170 pS in 150 mol/m 3 (mM) KC1), high ion selectivity (Pc~/PK = 0.015 -+ 0.01) and voltagedependent kinetics of this channel indicate that it is a type of maxi-K channel commonly found in animal cells but not previously detected in any plant cell.The current-voltage (I/V) characteristic of these channels was examined in drop-attached and in excised outside-out patches using the patch-clamp technique, over the unusually large voltage range of -250 to 200 mV. The I/V characteristic is nonlinear and shows saturation at extreme voltages; the current also saturates at high [K § In solutions with symmetrical KC1 concentrations the saturation behavior of the current is asymmetrical. The permeability of the channel depends on whether it is observed in excised or in drop-attached membrane patches.Here we investigate the main factors affecting the permeation of K + ions through this maxi-K channel. We present the first direct evidence for the importance of diffusion external to the pore in limiting ion flow through maxi-K channels. The data are consistent with an ion translocation mechanism whose current is limited (i) at high voltages by ion diffusion external to the pore and (ii) at high [K +] by the maximum transport rate of the channel. We fit the data to a diffusion-limited pore model in which the pore exhibits saturation described by Michaelis-Menten kinetics with a Km= 50 _+ 25 mol/m 3 and Gmax = 300 -+ 20 pS.

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“…After making a seal and verifying patch integrity, we excised the patch from the cell for freezing or labeling with antibodies. To protect the patch from dehydration during transfer from the recording medium, we covered the tip of the pipette with a sleeve of Teflon tubing (12 mm long; 1.6-mm inner diameter) (Quartararo and Barry, 1987). The sleeve, which had a constriction to prevent it from slipping, was slipped over the back of the pipette before inserting the pipette into the electrode holder.…”

Section: Patch Clampmentioning

confidence: 99%

The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy.

Ruknudin

Song

Sachs

1991

The Journal of Cell Biology

113

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Abstract. We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 t~m from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100/~m. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays.The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.

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A simple technique for transferring excised patches of membrane to different solutions for single channel measurements

Cited by 11 publications

References 11 publications

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta

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

The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy.

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