The effect of phosphatase inhibitors and agents increasing cyclic-AMP-dependent phosphorylation on calcium channel currents in cultured rat dorsal root ganglion neurones: interaction with the effect of G protein activation.

Abstract

Ca2+ channel currents have been recorded in cultured rat dorsal root ganglion neurones. The amplitude of IBa(GTP gamma S), recorded in the presence of GTP[ gamma S] (200 microM) in the patch pipette solution, is enhanced by external application of forskolin (10 microM), and there is an increase in the proportion of the rapidly activating component of the current. When forskolin (1 microM) is present in the bathing solution at the start of recording, or when 8-bromocyclic AMP (100 microM) is present in the patch pipette solution, the amplitude and rate of activation of IBa(GTP gamma S) are also increased compared to control IBa(GTP gamma S). The effect is mimicked by internal application of a 5 microM solution of a phosphopeptide fragment of inhibitor 1 (I1 PP), which inhibits phosphatase 1. The enhancement of IBa(GTP gamma S) caused by I1PP is not additive with that due to forskolin. Furthermore, the enhancement due to I1PP is reversibly lost when the holding potential is shifted from -80 mV to -30 mV, as was the enhancement due to forskolin and 8-bromocyclic AMP. I1PP also produced a less marked stimulation of the control Ca2+ channel current in the absence of G protein activation. The results suggest that phosphorylation regulates the interaction between calcium channels and G proteins in these neurones, and that phosphatase 1 is tonically active to dephosphorylate the relevant protein(s).