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. 2024 Sep 17;121(39):e2318900121. doi: 10.1073/pnas.2318900121

Fig. 1.

Fig. 1.

Regulation of IK,Ca in cardiomyocytes using optogenetics. (A) Schematic illustrating the dimerization of the CRY2 and CIBN optogenetic constructs at the plasma membrane and dephosphorylation of PIP2 via inositol-5-phosphatase (5P) activity to produce phosphatidylinositol 4-phosphate (PIP). (B) IK,Ca recorded in CHO cells using a voltage-ramp protocol prior to and during blue light exposure (blue arrow in the Left panel and black lines in the Right panel). (C) Current traces and the two-pulse voltage-clamp protocol used to isolate apamin-sensitive current activated by Ca2+ influx through L-type Ca2+ channels (LTCCs). The voltage-clamp protocol uses a prepulse to progressively induce Ca2+ influx via LTCCs from a holding potential of −55 mV, followed by a test pulse to monitor the apamin-sensitive IK,Ca. We recorded Ca2+ current (ICa) for each cell to measure the reversal potential (ECa). The test pulse was stepped to the observed ECa to minimize inward ICa. Specific inhibitors for transient outward K+ current (Ito), rapidly activating (IKr) and slowly activating delayed rectifier K+ currents (IKs), inward rectifier K+ currents (IK1), and Cl− currents were applied. The instantaneous outward K+ currents progressively increased depending on the Ca2+ influx. (D) Rabbit ventricular myocytes were transfected with optogenetic constructs containing inositol-5-phosphatase (5P, Left panel) compared to control (inactive phosphatase, 5P-Dead, Right panel) using magnetic nanoparticles. (E) The activation kinetics of IK,Ca was quantified compared to the total charge entered through LTCC during the prepulse (QCa in pC). Current traces together with insets showing activation kinetics of IK,Ca (red line) compared to the total charge entered through LTCCs during the prepulse (QCa in pC, blue line), before and after the blue light for rabbit ventricular myocytes transfected with optogenetic constructs containing inositol-5-phosphatase (5P, Left Panel) compared to control (inactive phosphatase, 5P-Dead, Right Panel). (F) IK,Ca was significantly blocked after blue light exposure compared to control cells. *P = 0.01.