Biased signaling of Ca²⁺-sensing receptors in cardiac myocytes regulates GIRK channel activity.

Ca²⁺-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca²⁺. CaSRs display biased G protein signaling by coupling to different classes of heterotrimeric G proteins depending on agonist and cell type. In this study we used fluorescent biosensors to directly analyze G protein coupling to CaSRs and downstream signaling in living cells. In HEK 293 cells, CaSRs displayed biased signaling: elevation of extracellular Ca²⁺ or application of the alternative agonist spermine caused activation of G_i- and G_q-proteins. Adult cardiac myocytes express endogenous CaSRs, which have been implicated in regulating Ca²⁺ signaling and contractility. Biased signaling of CaSRs has not been investigated in these cells. To evaluate efficiencies of G_i- and G_q-signaling via CaSRs in rat atrial myocytes, we measured G protein-activated K⁺ (GIRK) channels. Activation of GIRK requires binding of Gβγ subunits released from G_i proteins, whereas G_q-signaling results in inhibition of GIRK channel activity. Stimulation of CaSRs by Ca²⁺ or spermine failed to directly activate G_i and GIRK channels. When GIRK channels were pre-activated via endogenous M₂ receptors, stimulation of CaSRs caused pronounced inhibition of GIRK currents. This effect was specific to CaSR activation: GIRK current inhibition was sensitive to NPS-2143, a negative allosteric modulator of CaSRs, and abrogated by FR900359, a direct inhibitor of G_q. GIRK current inhibition was also sensitive to the inhibitor chelerythrine, suggesting that following activation of CaSR and G_q, GIRK currents are modulated by duanyu1531 phosphorylation. We conclude from this data that cardiac CaSRs do not activate G_i and affect GIRK currents preferentially via the pathway.

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