Equal sensitivity of Cav1.2 and Cav1.3 channels to the opposing modulations of PKA and PKG in mouse chromaffin cells

Mouse chromaffin cells (MCCs) express high densities of L-type Ca2+ channels (LTCCs), which control pacemaking activity and catecholamine secretion proportionally to their density of expression. In vivo phosphorylation of LTCCs by cAMP–PKAand cGMP–PKG, regulate LTCC gating in two opposing ways: the cAMP–PKA pathway potentiates while the cGMP–PKG cascade inhibits LTCCs. Despite this, no attempts have been made to answer three key questions related to the two Cav1 isoforms expressed in MCCs (Cav1.2 and Cav1.3): (i) how much are the twoCav1 channels basallymodulated by PKA and PKG?, (ii) to what extent canCav1.2 andCav1.3 be further regulated by PKA or PKGactivation?, and (iii) are the effects of both kinases cumulative when simultaneously active? Here, by comparing the size of L-type currents of wild-type (WT; Cav1.2+Cav1.3) and Cav1.3−/− KO (Cav1.2) MCCs, we provide new evidence that both PKA and PKG pathways affect Cav1.2 and Cav1.3 to the same extent either under basal conditions or induced stimulation. Inhibition of PKAbyH89 (5 μM) reduced the L-type current inWT and KO MCCs by∼60%,while inhibition of PKGby KT 5823 (1 μM) increased by∼40% the same current in both cell types. Given that Cav1.2 and Cav1.3 carry the same quantity of Ca2+ currents, this suggests equal sensitivity of Cav1.2 and Cav1.3 to the two basal modulatory pathways. Maximal stimulation of cAMP–PKA by forskolin (100 μM) and activation of cGMP–PKG by pCPT-cGMP (1mM) uncovered a∼25% increase of L-type currents in the first case and∼65% inhibition in the second case in bothWT and KO MCCs, suggesting equal sensitivity of Cav1.2 and Cav1.3 during maximal PKA or PKGstimulation. The effects of PKA and PKGwere cumulative and most evident when one pathway was activated and the other was inhibited. The two extreme combinations (PKA activation–PKG inhibition vs. PKG activation–PKA inhibition) varied the size of L-type currents by one order of magnitude (from 180% to 18% of control size). Taken together our data suggest that: (i) Cav1.2 and Cav1.3 are equally sensitive to PKA and PKG action under both basal conditions and maximal stimulation, and (ii) PKA and PKG act independently on both Cav1.2 and Cav1.3, producing cumulative effects when opposingly activated. These extreme Cav1 channel modulations may occur either during high-frequency sympathetic stimulation to sustain prolonged catecholamine release (maximal L-type current) or following activation of the NO–cGMP–PKG signalling pathway (minimal L-type current) to limit the steady release of catecholamines