Fabrication of patch pipets.

Patch clamping refers to a wide range of electrophysiological measurements, all of which have in common the use of patch pipets and the formation of gigaohm seals. The purpose of this unit is to describe the fabrication of patch pipets. The aspects of the pipet geometry that are important to different applications and the different procedures that have been found to most reliably and simply achieve these results are described. Parameters for glass selection are detailed in the beginning of the unit. Pulling patch and whole-cell pipets, elastomer coating, fire polishing, pipet filling, and pipet testing in an experimental setup are highlighted. Additional support protocols describe alternative ways to optimize pipet geometry and cleaning the glass before pulling. Considerations for noise and dynamic performance are emphasized as these two requirements for single-channel and whole-cell current measurements dictate how the pipets must be fabricated.

Origins of open-channel noise in the large potassium channel of sarcoplasmic reticulum.

Open-channel noise was studied in the large potassium channel of the sarcoplasmic reticulum (SR). Inside-out patches were excised directly from the SR of split skeletal muscle fibers of lobster, with lobster relaxing ringer (LRR) in bath and pipette. The power spectrum of open-channel noise is very low and approximately flat in the 100 Hz-10 kHz frequency range. At 20 degrees C, with an applied voltage of 50 mV, the mean single-channel current (i) is 9 pA (mean single-channel conductance = 180 pS) and the mean power spectral density 1.1 x 10(-29) A2/Hz. The latter increases nonlinearly with (i), showing a progressively steeper dependence as (i) increases. At 20 mV, the mean power spectral density is almost independent of (i) and approximately 1.4 times that of the Johnson noise calculated for the equivalent ideal resistor with zero net current; at 70 mV it increases approximately in proportion to (i)2. The mean power spectral density has a weak temperature dependence, very similar to that of (i), and both are well described by a Q10 of 1.3 throughout the range 3-40 degrees C. Discrete ion transport events are thought to account for a significant fraction of the measured open-channel noise, probably approximately 30-50% at 50 mV. Brief interruptions of the single-channel current, due either to blockage of the open channel by an extrinsic aqueous species, or to intrinsic conformational changes in the channel molecule itself, were a possible additional source of open-channel noise. Experiments in modified bathing solutions indicate, however, that open-channel noise is not affected by any of the identified aqueous species present in LRR. In particular, magnesium ions, the species thought most likely to cause brief blockages, and calcium and hydrogen ions, have no detectable effect. This channel's openings exhibit many brief closings and substrates, due to intrinsic gating of the channel. Unresolved brief full closings are calculated to make a negligible contribution (< 1%) to the measured power spectral density. The only significant source of noise due to band width-limited missed events is brief, frequent 80% substrates (mean duration 20 microseconds, mean frequency 1,000 s-1) which account for a small part of the measured power spectral density (approximately 14%, at 50 mV, 20 degrees C). We conclude that a large fraction of the measured open-channel noise results from intrinsic conductance fluctuations, with a corner frequency higher than the resolution of our recordings, in the range 10(4)-10(7) Hz.(ABSTRACT TRUNCATED AT 400 WORDS)

The use of quartz patch pipettes for low noise single channel recording.

Quartz has a dissipation factor of approximately 10(-4), which is an order of magnitude less than that of the best glasses previously used to fabricate patch pipettes; it's dielectric constant of 3.8 is also lower than that of other glasses. On the basis of these electrical characteristics it is expected that patch pipettes pulled from quartz tubing will produce significantly less noise than pipettes made from other glasses. Our work confirms these expectations and we describe theoretical and practical aspects of the use of quartz pipettes for single channel patch voltage clamp measurements. Methods for pulling quartz pipettes with a laser-based puller and coating them with low-loss elastomers are discussed, as are precautions that are necessary to achieve low noise recordings. We have shown that quartz pipettes can be pulled from tubing with outer diameter to inner diameter ratios as large as 3 and a method of applying heavy elastomer coatings all the way to the tip of pipettes is presented. Noise sources arising from the pipette and its holder are described theoretically, and it is shown that measured noise is in good agreement with such predictions. With low noise capacitive feedback electronics, small geometry holders, and thick-walled quartz pipettes coated with low-loss elastomers we have been routinely able to achieve noise of 100 fA rms or less in a 5-kHz bandwidth with real cell patches and a pipette immersion depth of approximately 2 mm. On occasion we have achieved noise as low as 60 fA rms in this bandwidth.

A method for exceptionally low noise single channel recordings.

We present a method whereby, with integrating electronics, quartz patch electrodes and a novel use of silicone oil, background noise levels as low as .083 pA RMS in a 5 kHz bandwidth (4-pole Butterworth filter) have been achieved in single channel patch clamp recordings. These approaches result in much higher signal to noise ratios for single channel recording than have previously been reported and should allow many investigators to significantly reduce noise at a constant bandwidth or to increase their recording bandwidths by several kHz.

Constructing a patch clamp setup.

It should be obvious that there are many ways to construct clamp set-ups that are either equivalent or sufficient for the experiments planned. The hardware and electronics can be obtained from several manufacturers, as can analysis software. What we have presented here are guidelines primarily meant to point a new experimenter in the right direction and, we hope, to guide more experienced investigators toward techniques that can improve the resolution of their measurements.

Glass technology for patch clamp electrodes.

Based on all of the properties of glass described here, it is obvious that no one glass can be recommended for all purposes and for all cells. Borosilicate glasses like 7760, 7052, and 7040 are good general purpose glasses for both single-channel and whole-cell recordings. They are good initial choices but, of course, must be checked for each cell type for problems associated with leaching of blockers, etc., from the glass. Corning 8161 is the best glass studied to date with respect to electrical and thermal properties but must be checked carefully for leachable components. If perforated-patch whole-cell recordings are to be used, 8161, KG-12, or some other high lead, low melting point glass are probably the best choices.

Planar bilayer membranes made from phospholipid monolayers form by a thinning process.

We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.

Electrical properties of sheep Purkinje strands. Electrical and chemical potentials in the clefts.

The impedence of sheep Purkinje strands, measured to 3-5 kHz, is interpreted with circuit models based on morphology. The strand is described as a one-dimensional electrical cable. Clefts between myocytes of the strand allow radial current to flow in parallel with current across the outer membrane. A lumped model of the clefts, in which all the cleft membrane is in series with 100 omega-cm2, fits only below 20 Hz. Two distributed models, pie and disk, fit at all frequencies with somewhat different (31%) luminal resistivities, but with similar membrane parameters. Series resistance representing the endothelial sheath is small. Simulations of voltage clamp experiments include measured linear parameters and nonlinear membrane channels, as well as radial variation of cleft concentration, membrane flux, voltage, and current. Cleft potential is drastically nonuniform when sodium current flows. Cleft potential is reasonably uniform when calcium and potassium currents flow, but the calcium and potassium concentrations change markedly, enough to turn off the calcium current, even if the calcium channel did not inactivate. We conclude that physiological current flows produce significant nonuniformities in electrochemical potentials in the clefts of this cardiac preparation.

Electrical models of excitation-contraction coupling and charge movement in skeletal muscle.

The consequences of ionic current flow from the T system to the sarcoplasmic reticulum (SR) of skeletal muscle are examined. The Appendix analyzes a simple model in which the conductance gx, linking T system and SR, is in series with a parallel resistor and capacitor having fixed values. The conductance gx is supposed to increase rapidly with depolarization and to decrease slowly with repolarization. Nonlinear transient currents computed from this model have some of the properties of gating currents produced by intramembrane charge movement. In particular, the integral of the transient current upon depolarization approximates that upon repolarization. Thus, equality of nonlinear charge movement can occur without intramembrane charge movement. A more complicated model is used in the text to fit the structure of skeletal muscle and other properties of its charge movement. Rectification is introduced into gx and the membrane conductance of the terminal cisternae to give asymmetry in the time-course of the transient currents and saturation in the curve relating charge movement to depolarization, respectively. The more complex model fits experimental data quite well if the longitudinal tubules of the sarcoplasmic reticulum are isolated from the terminal cisternae by a substantial resistance and if calcium release from the terminal cisternae is, for the most part, electrically silent. Specific experimental tests of the model are proposed, and the implications for excitation-contraction coupling are discussed.