Radioligand Binding

2.7.1 lodinated a-bungarotoxin(aBTX) binding

i) Crude membrane preparation

To determine total [^^^IJ-aBTX-binding to cell membranes, cells were washed once with PBS then scraped into cold PBS and pelleted by centrifugation at 14 OOOg for 20 minutes in a benchtop microfuge at 4°C. The cell pellet was resuspended in ice-cold lOmM potassium phosphate buffer (pH7.4) or Hanks Buffered Saline Solution (HBSS) containing lOug/ml each of leupeptin, apoprotinin and pepstatin. The crude membrane preparation was taken up 5 times through a 23 gauge needle and vortexed into a uniform suspension.

In Chapter 3.0, Section 3.1, crude membranes were incubated with lOnM

aBTX of specific activity >200 Ci/mmol (Amersham) in the presence or absence of lOmM carbamylcholine to determine non-specific binding. Samples were incubated in Eppendorf tubes for 2 hours at 4°C. Samples were washed 3 times with 1 ml PBS by brief centrifugation and resuspension. Bound [l^^I]-aBTX was determined by counting the Eppendorf tubes in a Wallac 1261 gamma counter.

For the remaining Results sections of this project, membranes were incubated for 2 hours at 4°C with l-2nM [^^^Ij-aBTX of specific activity >200 Ci/mmol (Amersham) with a final concentration of 0.5% bovine serum albumin (BSA). The presence of BSA reduced the non-specific binding of [^^^IJ-aBTX to the glass fibre filter. This was usually added with the [^^^I]-aBTX to enable simple protein assays of the cell samples. Non-specific binding was determined by the addition to duplicate or triplicate tubes of: 100|iM methyllycaconitine (MLA, Research Biochem icals International) for samples transfected with a 7 , or 500mM carbamylcholine plus 500mM nicotine (both from Sigma) for samples transfected

with a8 or the muscle nAChR. Samples were harvested using a Brandel Harvester

(Model M-36, Semat, U.K.) onto GF-A glass-fibre filters pre-soaked for 2 hours in 0.5% polyethylenimine (PEI) (Sigma) with five or six 3ml washes of ice-cold lOmM phosphate buffer. Filters were counted in a Wallac 1261 gamma counter. Typically, the glass fibre filters would collect -0.1-0.3% of the [^^^IJ-aBTX counts included in the assay irrespective of the presence of cellular material.

For saturation binding studies of the clonal SH-SY5Y-a7#7 cell line, increasing concentrations of [l^^IJ-aBTX were used (2pM-6nM). Curves for equilibrium binding were fitted by equally weighted least squares (CVFIT program, David Colquhoun, University College London). The calculated Hill coefficients did not differ significantly from 1, so data were refitted using the Hill-Langmuir equation (with »H=1) in order to estimate the equilibrium constants for ocBTX-binding.

ii) Intact cell suspension

To determine [^^^IJ-aBTX-binding to cell surface membranes, cells were rinsed twice with pre-warmed Hanks Buffered Saline Solution (HBSS) and gently scraped or titurated from the culture dish into 1ml HBSS. HBSS was found to be more osmotically balanced than standard PBS (Dulbecco's A) which proved more effective at keeping the cells intact over longer periods. Prior to addition to the assay tubes, the cells were gently separated into a single-cell suspension by pipetting 3 times with a 1ml pipette tip. Intact cells were incubated with l-2nM [^^^IJ-aBTX in HBSS containing 0.5% BSA for either 1 hour at 37°C, or at room temperature for 1.5-2.0 hours. Non-specific binding was determined by the addition to duplicate or triplicate tubes of: lOOp-M MLA for samples transfected with a l , or 500mM carbamylcholine plus 500mM nicotine for samples transfected with a8 or the muscle nAChR. The

harvesting and washing of the samples was performed as described in Section 2.7. l(i).

Hi) Cell monlayers

Intact cells were labelled with 5-20 nM [^^^I]-aBTX in their usual culture media for 1 hour at 37°C. Cells were rinsed four times with PBS or HBSS and solubilized in lysis buffer containing protease inhibitors for 1-2 hours on ice. Samples were centrifuged at full speed in a benchtop microfuge for 1 0 minutes to pellet any unsolubilised

material. The supernatant was transferred to a fresh tube and subsequently used for sucrose gradient centrifugation or immunoprécipitation of bound [i^^I]-aBTX.

2.7.2 Tritiated radioligand binding

i) [^H]-epibatidine

[^HJ-Epibatidine binding was performed with cell homogenates. Crude membrane suspensions were prepared as for the [l^^I]-aBTX binding assay (see Section 2.7.Iz). Membranes were incubated for 2 hours on ice with 3nM [^HJ-epibatidine of specific activity 53 Ci/mmol (Amersham) in HBSS containing lOug/ml each of leupeptin, apoprotinin and pepstatin. Non-specific binding was determined by the addition of 500|iM nicotine and 500|iM carbamylcholine (both from Sigma) to duplicate or triplicate samples. Samples were harvested using a Brandel Harvester onto GF-B glass fibre filters pre-soaked for 2 hours in 0.5% PEI and washed four times with ~3mls cold potassium phosphate buffer (pH 7.4). The filters were plucked from the filter sheets and transferred to scintillation vials. 5mls of 'Ready Safe' liquid scintillation cocktail (Beckman) was added to the vials and shaken overnight. Samples were counted in a Beckman LS 6500 Scintillation Counter.

For saturation binding studies of the GH^Ci-aS transfected cells, increasing concentrations of [^H]-Epibatidine were used (5pM-10nM). Curves for equilibrium binding were fitted by equally weighted least squares (CVFIT program, David Colquhoun, University College London).

ii) pH]-GR65630

The binding of the specific 5-HTg antagonist, GR65630, was performed on cell homogenates as above. Membranes were incubated for 2 hours at 4°C with InM [^H]-GR65630 (specific activity 61.4 Ci/mmol, NEN) in the presence or absence of 100|iM 5-hydroxytryptamine (Sigma) to determine non-specific binding. Samples were harvested using a Brandel Harvester onto GF-B glass fibre filters pre-soaked for 2 hours in 0.5% PEI and washed four times with ~3mls cold lOmM potassium phosphate buffer (pH 7.4).