Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras

Influx of calcium through voltage-dependent calcium channels (VDCCs) has been implicated in the processes of cell growth and differentiation. Various signalling proteins, including nerve growth factor (NGF), p21-ras and src tyrosine kinases, have been suggested to have a role in the regulation of neuronal VDCCs. Using the whole-cell patch-clamp technique we have investigated the role of endogenous p21-ras in the regulation of VDCCs in primary cultured dorsal root ganglion (DRG) neurons obtained from neonatal rats. Neutralization of endogenous p21-ras by microinjection of p21-ras antibody (Y13-259) reduced the maximum peak barium current, I(max), whereas microinjection of oncogenic p21-K-ras increased the current. Thus, endogenous p21-ras is involved in the tonic regulation of calcium currents in these cells. Intracellular application of a phosphopeptide, Trk 490, which prevents the binding of the adaptor protein shc to the activated NGF receptor, so blocking p21-ras activation, reduced I(max). Similarly, deprivation of NGF by overnight incubation in NGF-free medium also reduced I(max). Together, these results suggest that NGF receptor tyrosine kinase activation of p21-ras is likely to be involved in the tonic regulation of VDCCs in DRG neurons. Deprivation of NGF combined with microinjection of p21-ras antibody (Y13-259), however, caused an even greater reduction of I(max). Thus, NGF activation can only partially explain the regulation of these currents by endogenous p21-ras. Src tyrosine kinases have been suggested to activate p21-ras. In DRG neurons, microinjection of purified src tyrosine kinase, pp60c-src, increased I(max) in these cells. However, co-microinjection of pp60c-src with Y13-259 antibody prevented the increase in I(max), implying that pp60c-src can also regulate calcium currents via the activation of endogenous p21-ras. Further support for the involvement of tyrosine kinases in VDCC regulation was provided by the application of the general tyrosine kinase inhibitor, genistein, which also reduced I(max). Thus, VDCCs in rat DRG neurons appear to be tonically up-regulated by endogenous p21-ras. This effect appears largely to involve NGF receptor tyrosine kinase activation of p21-ras. In addition, src tyrosine kinase may also regulate VDCCs, possibly via p21-ras.