Whole-cell patch clamping

Pharmacological agents such as NAADP that dissolve readily in water are often unable to diffuse across the plasma membrane. This property is useful for an intracellular signalling molecule, however observing the effect of exogenous NAADP is therefore far from straight forward. In order to deliver NAADP directly to the cytoplasm of both PASMCs and HEK293 cells the whole-cell configuration of the patch-clamp technique was used. The key characteristic of this technique is the diffusional continuity of the patch pipette solution and the cell cytoplasm (Park et al., 2002).

2.6.1 Preparation and filling of patch pipettes

In this thesis, whole-cell patch-clamping was used to apply the pharmacological agent of interest by intracellular dialysis within pipette solution of the following composition: 140 mM KCl; 10 mM HEPES; 1 mM MgCl2; 5 µM Fura-2 free acid,

corrected to pH 7.4 with NaOH. The pipette solution was ‘loaded’ into a glass electrode known as a patch pipette. Patch-pipettes (2-3 MΩ resistance) were

generated by ‘pulling’ filamented borosilicate glass capillaries (1.5 mm O.D., 0.86 mm I.D.; Harvard Apparatus, UK) using a pipette puller (pp-830, Narishige, Japan). The pp-830 pipette puller uses a two stage pull technique to produce pipettes with a consistent pipette resistance. Firstly, the glass capillary is fixed in position by screws above and below the nichrome heating coil, with the upper part fixed to the frame of the puller and the lower part attached to weights. The heating coil is then switched on to the first heat setting which heats the capillary at a focal position. As this part of the capillary heats up, the glass becomes molten and the glass is partially pulled apart by gravity pulling the weighted lower section down until it makes contact with a step. This first pull is typically over a length of 7-10 mm and produces a diameter of approximately 200 µm (Hamill et al., 1981). The step physically stops the first stage

in the pulling process and also switches off the heating coil thus allowing both the coil and capillary glass to cool down. The coil is then re-centred with respect to the capillary’s new centre, the step is removed and the coil switched on to the second, lower heating setting. This heat also focally softens the glass and therefore allows the

weights to pull apart the glass, leaving two patch-pipettes with tips consisting of an aperture of approximately 1 µm (Hamill et al., 1981).

The filled patch pipettes were mounted onto the electrode attached to the headstage. Once mounted, the pipette solution was in electrical contact with the head stage via an Ag/AgCl wire which sits within the pipette. The headstage is attached to the patch-clamp amplifier, and the amplifier kept in electrical contact with the extracellular bath solution via an Ag/AgCl bath electrode.

2.6.2 Generation of a seal and delivery of pharmacological agents

Once a cell had been identified for patching, the patch pipette was positioned above the cell of interest using a piezoelectric micromanipulator (PCS 5000 series, EXFO Burleigh, Canada). The piezoelectric micromanipulator allows smooth and stable positioning of the patch pipette within three planes. The system uses ceramic actuators that expand under the application of increasing voltages. Before a seal can be made with the cell of interest, the junction potential must be accounted for. The junction potential develops when two solutions of different ionic compositions meet at an interface. In this example, once the pipette is lowered into the experimental chamber, the bath solution and the pipette solution share an interface at the pipette aperture. This causes a junction potential to develop due to the differences in concentration and mobility of ions between the solutions. This potential can be offset by adjusting the Vref on the amplifier which uses an integrator to keep the current at

zero. Before making a seal the amplifier must first be set to voltage-clamp mode (V = 0 mV) with a 5 mV ‘test’ pulse applied with a frequency of 50 Hz and a duration of 10 ms. The pulse produces a current step with a square waveform which is observed on an oscilloscope (Fig. 2.5). The patch pipette is then lowered gradually using the micromanipulator until the patch pipette is in contact with the cell membrane. Contact with the cell can be determined using the oscilloscope whereby the increase in resistance results in a decrease in the observed current step. At this juncture, negative pressure can then be applied to the system. Negative pressure could be applied to the pipette via a length of silicon tubing that was attached to the electrode holder. The other end of the tubing was attached to a port in a 3-way stopcock. A plastic syringe was attached to another port of the stopcock to generate the negative

pressure whilst the remaining port was left free in order to release pressure from the otherwise closed system.

Fig. 2.5. Schematic representation of the process of intracellular dialysis of pharmacological agents via the whole-cell configuration of the patch-clamp technique

A. The upper panel shows the positioning of the patch pipette above the cell. Lower panel shows the corresponding trace shown on the oscilloscope after the application of a voltage step. B. The upper panel shows the patch pipette now lowered so that it is contact with the cell plasma membrane, whilst the lower panel shows the resultant decrease in the voltage step as a result of an increased resistance.

C. Application of slight negative pressure generates a gigaseal shown in the upper panel. The lower panel shows the resultant oscilloscope trace after capacitance compensation. D. The upper panel represents the whole-cell configuration. Further negative pressure results in the removal of the patch of membrane immediately beneath the patch pipette. The lower panel shows the oscilloscope trace with whole-cell configuration producing characteristic capacitance transients.

Application of negative pressure results in an improved seal and increased electrode resistance resulting in a further reduction in the current step observed on the oscilloscope. This was monitored until a gigaohm (GΩ) seal was obtained with

additional negative pressure applied if required. With a 10 GΩ seal the pipette and

the membrane will be less than 1 nm apart (Hamill et al., 1981). After forming a GΩ

seal, capacity transients caused by stray pipette capacitance to the bath, are compensated for by adjusting the fast and slow capacitance compensation facilities on the integrator unit of the amplifier. Further suction was then applied in order to rupture the patch of membrane covered by the pipette’s aperture. Rupture of the patch is indicated by the sudden appearance of large capacity transients due to whole- cell capacitance. This is termed ‘break-in’. Upon break-in the NAADP within the

pipette solution is able to dialyse into the cell and the resultant changes in the intracellular Ca2+ concentration was viewed using Fura-2 fluorescence ratio imaging. For all experiments involving the whole-cell configuration of the patch-clamp technique, the seal resistance was ≥ 2 GΩ, and the series resistance and pipette

resistance were ≤ 10 MΩ and ≤ 3 MΩ respectively.

NAADP and IP3 were delivered to individual cells using the whole-cell

configuration of the patch-clamp technique and in current-clamp mode (PASMC; I = 0) or voltage-clamp mode (HEK293 cells; V = -40 mV), using a CV 203BU headstage (Axon Instruments, Molecular Devices, USA) attached to an axopatch 200B integrating patch-clamp unit (Axon Instruments, Molecular Devices, USA).

2.6.3 Extracellular application of pharmacological agents

In Ca2+ imaging experiments in both PASMC and HEK293 cells, caffeine or carbachol (CCh) were added towards the end of the record in order to determine whether the cells were responsive to intracellular Ca2+ release via RyRs or IP3Rs

respectively. As both caffeine and CCh are cell membrane permeant, these compounds were applied extracellularly. Also, is some of the experiments within this thesis, cells were pre-incubated with the antagonists, bafilomycin A1, thapsigargin, dantrolene and Ned-19. These compounds are also cell permeable and were therefore applied extracellularly.