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

Key points Cav1.2 and Cav1.3 L-type calcium channels are highly expressed in rat and mouse chromaffin cells. Beside shaping and pacemaking action potential trains, they regulate vesicle exocytosis and endocytosis. L-type channels are opposingly regulated by the cAMPPKA and cGMPPKG pathways and their Ca2+ current can undergo marked up and down changes. To date, most of the reported findings on L-type channel modulation derive from the cardiac Cav1.2 isoform. Here, using wild-type and Cav1.3 knock out (KO) mouse chromaffin cells we show that, like Cav1.2, Cav1.3 channels are effectively modulated by PKA and PKG at basal conditions and during maximal PKA/PKG stimulation. The extent of modulation is nearly equal for both Cav1 channel isoforms. PKA and PKG pathways act independently on Cav1.2 and Cav1.3, producing cumulative effects that are mostly visible when activating PKA and inhibiting PKG, or vice versa. Under these conditions the L-type Ca2+ current can undergo changes of one order of magnitude. These extreme Cav1 channel modulations are likely to occur during different physiological conditions of the adrenal gland: fight-or-flight response vs. relaxed states. Abstract 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 cAMPPKA and cGMPPKG, regulate LTCC gating in two opposing ways: the cAMPPKA pathway potentiates while the cGMPPKG 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 two Cav1 channels basally modulated by PKA and PKG?, (ii) to what extent can Cav1.2 and Cav1.3 be further regulated by PKA or PKG activation?, 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 PKA by H89 (5 mu m) reduced the L-type current in WT and KO MCCs by similar to 60%, while inhibition of PKG by KT 5823 (1 mu m) increased by similar to 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 cAMPPKA by forskolin (100 mu m) and activation of cGMPPKG by pCPT-cGMP (1 mm) uncovered a 25% increase of L-type currents in the first case and 65% inhibition in the second case in both WT and KO MCCs, suggesting equal sensitivity of Cav1.2 and Cav1.3 during maximal PKA or PKG stimulation. The effects of PKA and PKG were cumulative and most evident when one pathway was activated and the other was inhibited. The two extreme combinations (PKA activationPKG inhibition vs. PKG activationPKA 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 NOcGMPPKG signalling pathway (minimal L-type current) to limit the steady release of catecholamines.