G_q/11 G protein-coupled receptors stimulate PIP₂ hydrolysis. This can be monitored at the single-cell level by translocation of the green fluorescent protein-tagged Pleckstrin Homology domain of phospholipase C δ (GFP-PLCδ-PH; Stauffer et al. 1998). We have used this method to monitor PIP₂ hydrolysis in dissociated sympathetic neurons in parallel with voltage-clamp measurements of agonist-induced M-current (I_M) inhibition.

GFP-PLCδ-PH was expressed in superior cervical ganglion neurons (SCGs) from 17-day-old rats (killed humanely: Home Office Schedule 1 procedure) via intranuclear injection of a cDNA plasmid. One day after injection, whole-cell M-currents were recorded using perforated-patch electrodes, and localisation of GFP-PLCδ-PH monitored via fluorescence microscopy. At rest, GFP-PLCδPH was located preferentially at the plasma membrane. Application of either the muscarinic acetylcholine receptor agonist oxotremorine M (oxo M; 10 µM) or bradykinin (BK; 100 nM) induced reversible translocation of GFP-PLCδ-PH from membrane to cytosol, in parallel with I_M inhibition. The PI3/4-kinase inhibitor wortmannin (20 µM) prolonged reversal of both.

Over-expression of the neuronal calcium sensor protein 1 (NCS-1; Burgoyne & Weiss, 2001) reduced BK-induced inhibition of I_M 2 days after injection (100 nM BK) from 43.0 ± 9.0 % (mean ± S.E.M., n = 5) in control cells to 14.1 ± 4.4 % (n = 6). The muscarinic inhibition of I_M (10 µM oxo M) was unaltered (66.0 ± 9.1 % (n = 5) – control, and 57.2 ± 7.8 % (n = 6) – NCS-1).

Oxo M- and BK-induced translocation of GFP-PLCδ-PH were measured as a rise in cytosolic fluorescence intensity (CFI). In the control group, the rises in CFI induced by both agonists were almost equal, the average BK-induced rise being 98 % of that induced by oxo M. The amplitude of the oxo M-induced rise in CFI was unchanged by NCS-1 overexpression. However, the BK-induced rise was significantly reduced.

These data extend recent electrophysiological observations relating PIP₂ turnover with I_M inhibition (Suh & Hille, 2002). Our results also indicate that NCS-1 reduces the sensitivity of I_M inhibition to BK by reducing the loss of PIP₂ from the membrane during agonist stimulation. We suggest that this is via an enhancement of PIP₂ synthesis, through increased PI4-kinase activity (Koizumi et al. 2002).

This work was supported by The Wellcome Trust and the Medical Research Council. We thank Dr J.L.Weiss (University of Sheffield) for the NCS-1 cDNA and Dr T. Meyer (Duke University) for the GFP-PLCδ-PH.