PMA counteracts G protein actions on Ca V 2.2 channels in rat sympathetic neurons Alfonso F. Dı ´az-Ca ´rdenas a,b , Isabel Arenas a , David E. Garcı ´a a, * a Departamento de Fisiologı ´a, Facultad de Medicina, Universidad Nacional Auto ´ noma de Me ´xico, Av. Universidad 3000, Ciudad Universitaria, Delegacio ´ n Coyoaca ´ n. C.P. 04510, Apdo. Postal 70250, Me ´xico, D.F., Me ´xico b Departamento de Psicologı ´a, Universidad Auto ´ noma de Puebla, C.P. 72000, Puebla, Me ´xico Received 15 November 2007, and in revised form 19 January 2008 Available online 23 February 2008 Abstract Protein kinase C (PKC)-induced phosphorylation and G protein-mediated inhibition of Ca V 2.2 N-type Ca 2+ channels counteract exerting opposing modulatory responses at the channel level. At present, the most striking question remaining is whether prominent enhancement of the Ca 2+ current (I Ca ) observed under PKC activation arises from relief of G-protein tonic inhibition. Here, by using patch-clamp methods in superior cervical ganglion (SCG) neurons of rat, we show the following: First, that PKC activation by phorbol- 12-myristate-13-acetate (PMA) not only counteracts mutually with noradrenaline (NA) and GTPcS-induced I Ca inhibition, but also reverses current inhibition by Gbc subunits over-expression. Second, that PMA increases I Ca beyond the enhancement expected by sole removal of the G protein-mediated tonic inhibition. Accordingly, PMA increases conductance through N-type Ca 2+ channels, unlike the G protein inhibitor GDPbS. Together, our results support that PMA-induced phosphorylation produces changes in I Ca that cannot be accounted for by prevention of G protein inhibition. They may have important implications in reinterpretation of existing data with PMA. Furthermore, counteracting modulation of ion channels and reversibility within a short time frame are better support for a dynamic system with short-term adaptive responses. Ó 2008 Elsevier Inc. All rights reserved. Keywords: G-protein; Gbc; Ca 2+ channels; Phorbol esters; PMA; Phosphorylation PMA is a well-known and widely used phorbol ester with proven ability to activate protein kinase C in superior cervical ganglion (SCG) neurons when employed at a 500- nM concentration [1–5]. It has long been reported that in rat sympathetic neurons PKC enhances Ca 2+ channel cur- rents by relieving G protein tonic inhibition in the absence of neurotransmitter or induced by G protein activation with GTPcS [6,1,7,8]. Interestingly, Zong and Lux [9] reported an augmentation of Ca 2+ channel currents in response to G protein activation by GTPcS as occurring within minutes, during which channels are supposedly inhibited by the activated G proteins. PKC activation has been proposed as one of the intra- cellular mechanisms mediating certain neurotransmitter- induced Ca 2+ channel activation, at least in some prepara- tions [2,10]. In general, it appears that sympathetic neurons utilize PKC to enhance Ca 2+ channel activities, whereas sensory neurons use PKC to inhibit these channels [2]. Thus, activation of PKC increases Ca 2+ channel currents in Aplysia neurons [11], frog [12], rat sympathetic neurons [1], and rat central neurons [1,13]. However, a discrepancy exists regarding the interaction of phosphorylation and G proteins on Ca 2+ channels in sympathetic neurons. Chen and Penington [14] proposed that the order of application determines the interaction between phorbol ester-induced phosphorylation and GTPcS. Barrett and Rittenhouse [4] reported that activated PKC is unable to reverse GTPcS- induced inhibition and concluded that phosphorylation by PKC exerts no effect on whole-cell N-type currents 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.01.030 * Corresponding author. Fax: +52 55 5623 2241. E-mail address: erasmo@servidor.unam.mx (D.E. Garcı ´a). www.elsevier.com/locate/yabbi ABB Available online at www.sciencedirect.com Archives of Biochemistry and Biophysics 473 (2008) 1–7