Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes

Abstract

We examined the role of the Ca2+‐regulated protein phosphatase calcineurin in controlling Ca2+ signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch‐clamp technique. Cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in cells loaded with the fluorescent Ca2+ indicators fluo‐4 or fura‐2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 nm) or the calcineurin auto‐inhibitory peptide (CiP, 100 μM), increased the amplitude and rate of decay of the evoked [Ca2+]i transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 nm) and 100 μM CiP on the [Ca2+]i transient and AP were not additive. Calcineurin inhibition did not modify the K+ currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca2+ current (ICa) and the evoked calcium transient normalized to the ICa. Calcium sparks, which underlie the [Ca2+]i transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co‐localize near the T‐tubules of ventricular myocytes. We propose that calcineurin and PKA are co‐localized to control Ca2+ influx through calcium channels and calcium release through ryanodine receptors.

Cyclosporin and Timothy syndrome increase mode 2 gating of CaV1.2 calcium channels through aberrant phosphorylation of S6 helices

“…Two human neurological disorders are associated with calcium cytotoxicity, which could result from a gain of function by calciumselective channels, but their relationship to mode 2 gating has not been investigated. One disorder is the neurotoxic effects of cyclosporin, an inhibitor of the calcium͞calmodulin-dependent protein phosphatase IIB calcineurin (11), which regulates transcription in lymphocytes (12) but has also been reported to inhibit dihydropyridine-sensitive calcium channels (13)(14)(15). Cyclosporin is given to transplant patients to inhibit the immune response and prevent rejection of the transplanted tissue; however, chronic cyclosporin use has unexplained neurotoxic side effects that could result in part from disinhibition of calcium channels (16).…”

Selective inhibition of calcineurin-NFAT signaling by blocking protein–protein interaction with small organic molecules

“…The conventional method of blocking calcineurin-NFAT signaling is to apply the immunosuppressive compounds cyclosporin A (CsA) and FK506, which, in the form of CsA-cyclophilin or FK506-FKBP12 complexes, inhibit the enzymatic activity of calcineurin toward all its physiological substrates (33). However, calcineurin employs a range of targeting mechanisms (1,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) that offer conceptually novel possibilities for disrupting calcineurinsubstrate signaling. In particular, a protein-protein interaction of calcineurin with NFAT-family proteins controls the efficiency of NFAT dephosphorylation in vitro and in cells (1,16,44,45).…”

Regulation of Renal Potassium Channels by Protein Kinases and Phosphatases

“…However, it has been reported that the target phosphorylation site which was dephosphorylated by CaN is mainly mediated by PKA (Santana et al, 2002). This report demonstrated that the functional coupling of CaN and PKA modulated Ca 2+ release in ventricular myocytes (Santana et al, 2002). It has also been demonstrated that Na + -K + www.intechopen.com pump at the basolateral membrane of kidney tubular epithelia was inhibited by CyA (Tumlin & Sands, 1993) and stimulated by PKA (Carranza et al, 1998).…”

“…The above data suggest that CaMKII and CaN are coupled mediators in modulating the ATPregulated K + channels. However, it has been reported that the target phosphorylation site which was dephosphorylated by CaN is mainly mediated by PKA (Santana et al, 2002). This report demonstrated that the functional coupling of CaN and PKA modulated Ca 2+ release in ventricular myocytes (Santana et al, 2002).…”