Emerging Evidence for cAMP-calcium Cross Talk in Heart Atrial Nanodomains Where IP ₃ -Evoked Calcium Release Stimulates Adenylyl Cyclases

Figures

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  Figure 1.. Co-localisation of IP3 receptors with calcium-stimulated adenyl cyclases identified by immunocytochemistry. A: An atrial myocyte in which IP3R2s are labelled red while AC8 is labelled green. B: The cellular location of these two proteins at higher magnification. C: A plot of intensity of red and green fluorescence against distance across the cell at the line at right angles to the long axis of the cell shown in panel B. D: An atrial myocyte in which IP3R2s are again labelled red while AC1 is labelled green. E: The cellular location of IP3R2 and AC1 at higher magnification, while F shows a plot of intensity of red and green fluorescence against distance across the cell at the line at right angles to the long axis of the cell shown in panel E. Note that there was colocalisation of IP3R2 and AC8, while AC1 appeared to be located on the cytosolic side of junctional SR. From Capel et al. (2021).

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  Figure 2.. Effects of IP3-evoked calcium release on calcium transients (CaT) depend on adenylyl cyclases and PKA. A plot of changes in amplitude of calcium transients (CaT) accompanying action potentials (stimulation frequency, 1 Hz) measured as fluo-5 fluorescence following intracellular photorelease of IP3 from a caged compound. In the absence of drugs photorelease of IP3 led to a progressive increase in CaT amplitude over the 3 min time period shown. These effects of photoreleased IP3 were suppressed when adenylyl cyclases were inhibited by MDL 12-330A (3μmol/L), and when PKA was inhibited by H89 (1μmol/L). The time control for changes in CaT amplitude in the absence of photorelease of IP3 showed a small progressive decline. The increase in CaT amplitude following photorelease of IP3 seemed little if at all affected by inhibition of eNOS by L-NAME or by inhibition of guanylyl cyclase by ODQ, showing that NO signaling seemed not to be a major contributor to the changes in CaT amplitude that resulted from IP3-evoked calcium release from the SR. From Capel et al. (2021).

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  Figure 3.. Schemes showing possible mechanisms of action of IP3-evoked calcium release (a) Scheme showing location of IP3 receptors (IP3R) in junctional SR, together with proteins relevant to the newly proposed signaling mechanism. IP3-evoked calcium release from SR stimulates calcium-activated adenylyl cyclases (AC8 and AC1) leading to production of cAMP and PKA, which in turn phosphorylates target proteins, L-type calcium channels (LTCC), ryanodine receptors (RyR) and phospholamban (PLB) associated with sarcoplasmic-endoplasmic reticulum calcium ATP-ase (SERCA). IP3 is produced in response to stimulation of a G-protein coupled receptor (GPCR) leading to splitting of PIP2 into diacylglycerol and IP3. AC8 is shown anchored to the surface membrane and to junctional SR since current immunocytochemistry with light microscopy does not allow us to distinguish between a location in surface membrane caveolae or SR. This designation is intended to emphasise that AC8 shows colocalisation with IP3Rs (as shown in Figure 1), so that if AC8 were in caveolae then presumably this component of the surface membrane must be in very close proximity to the IP3Rs in the junctional SR. AC1 appears to be anchored…

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  Figure 4.. SAN timing mechanisms. (a): Timing mechanism dependent on ionic currents in the surface membrane (‘Membrane Clock’). Activation of potassium channels by the SAN action potential leads to repolarization, but once the most negative potential is achieved de-activation removes their hyperpolarizing influence thus contributing to slow depolarization. Potassium channel currents include IKr (through ERG channel protein) and IKs (carried by KCNQ1 with KCNE1, also known as KVLQT1 and minK). HCN channels and sustained inward current (through Cav1.3 plus another unknown protein) further contribute depolarizing influences to the slow depolarization. Activation of calcium current through CaV1.3 channels also adds to the later stages of the slow depolarization, which then triggers the action potential upstroke via activation of a combination of CaV1.3 and CaV1.2. The cycle then repeats itself. Note that SAN cells, unlike atrial myocytes, lack the stabilising influence of IK1. Electrogenic calcium extrusion via NCX can also contribute to the depolarization, with the amplitude driven by the magnitude of changes in sub-sarcolemmal calcium concentration (see text and Capel and Terrar, 2015). (b):…

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  Figure 5.. Targets for cAMP and PKA in SAN following activation of AC1 and AC8 by IP3-evoked calcium release. This scheme is analogous to that shown in Figure 3(a) to represent IP3 signaling in the atria, but the proposed target proteins in the surface membrane are more diverse including HCN channels directly regulated by cAMP as well as a variety of surface membrane channel proteins phosphorylated by PKA. The targets for PKA again also include the SR proteins, PLB/SERCA and RyR, so that the scheme includes principal components of both the membrane clock and the SR calcium clock shown in Figure 4.

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  Figure 6.. Possible effects of SR calcium release via RyR on surface membrane ionic currents controlling membrane clock in SAN. Scheme building on observed effects of SR calcium release via RyR on L-type calcium currents (Collins and Terrar, 2012) through CaV1.2 and mediated by calcium stimulated adenylyl cyclases (AC8 and AC1). In SAN node, evidence also supports effects on HCN via direct effects of cAMP, and is likely also to involve other PKA targets such as CaV1.3, sustained inward current and potassium channels (see text). For simplicity in this scheme AC8 and AC1 are both shown close to the surface membrane, although it should be noted that the observations in Figure 1 show that AC1 appears to be on the intracellular side of junctional SR, while the location of AC8 appears to overlap with junctional SR.