Relationship between Membrane Phosphatidylinositol-4,5-Bisphosphate and Receptor-Mediated Inhibition of Native Neuronal M Channels

Abstract

The relationship between receptor-induced membrane phosphatidylinositol-4′5′-bisphosphate (PIP 2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP 2/inositol 1,4,5-trisphosphate (IP 3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCδ-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCδ-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCδ-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP 2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP 3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP 2] were estimated at 261 μ m(95% confidence limit; 192-381 μ m), rising to 693 μ m(417-1153 μ m) in neurons overexpressing PI5-K. Changes in membrane [PIP 2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP 2(Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP 2. The effects of bradykinin require additional components of action, which might involve IP 3-induced Ca 2+release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP 2 synthesis by Ca 2+-dependent activation of NCS-1.