Fine tuning by protein kinases of Ca_V1.2 channel current in rat tail artery myocytes

Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular Ca_V1.2 channel current (I_Ca1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate I_Ca1.2 and disclose any unexpected modulation of Ca_V1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced I_Ca1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The β-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased I_Ca1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced I_Ca1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased I_Ca1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated I_Ca1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Gö6976 markedly reduced basal I_Ca1.2 and, similarly to the PKCδ (rottlerin) and PKCε translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited I_Ca1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Gö6976 appeared to block directly Ca_V1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α_1C subunit in a region involved in Ca_V1.2 channel inactivation, forming an interaction network comparable to that of Ca_V1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.