Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes

Abstract

Small-conductance Ca²⁺-activated K⁺ (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca²⁺ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca²⁺ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca²⁺ microdomains necessary for SK channel activation. SK currents coupled with Ca²⁺ influx via L-type Ca²⁺ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca²⁺ store, suggesting that LTCCs provide the immediate Ca²⁺ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca_v1.2 Ca²⁺ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca_v1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca²⁺ signaling.

Figure 1

Apamin-sensitive SK currents in rabbit ventricular myocytes. (A) Representative current traces elicited from a holding potential of −55 mV using a family of voltage steps from −120 mV to +60 mV at 10 mV increments with 500 ms durations. Current traces shown are recorded in control (black traces, left panels), after 100 pM (blue traces), 10 nM (red traces), and 100 nM (green traces) of apamin (middle panels). Current traces in the right panels are apamin-sensitive currents obtained from digital subtraction. (B and C) Summary data for the current-voltage relationship of apamin-sensitive SK currents recorded from epicardial (Epi-) (n = 5) and endocardial (Endo-) (n = 7) ventricular myocytes.

Figure 2

Apamin-sensitive outward currents elicited using two-pulse voltage-clamp protocols. (A) Representative current traces elicited from a two-pulse voltage-clamp protocol used to isolate apamin-sensitive current activated by Ca²⁺ influx through L-type Ca²⁺ channels (LTCCs). 50 μM BAPTA was included in the pipette solution. A diagram depicting the two-pulse voltage-clamp protocol is shown in the inset. (B) Current traces elicited using a voltage-clamp protocol to elicit Ca²⁺ currents for the determination of E_Ca. A diagram depicting the voltage-clamp protocol is shown in the inset. (C and D) The outward SK currents in the absence (C) and presence of 100 nM apamin (D). The SK currents were activated by Ca²⁺ influx through LTCCs under conditions with 50 μM BAPTA in the pipette solution using the protocol shown in (A). The insets show activation kinetics of the outward K⁺ currents (in pA, open symbols) compared to the total charge entered through LTCCs during the prepulse (Q_Ca in pC, closed symbols).

Figure 3

Effects of BAPTA compared to EGTA on the activation of SK currents. (A and B) Ca²⁺ influx through LTCCs does not activate SK currents when the pipette solution contains 10 mM EGTA (A) or 10 mM BAPTA (B). (C and D). SK currents activated by Ca²⁺ influx through LTCCs when 50 µM EGTA (C) or 50 µM BAPTA (D) was included in the pipette solution. The insets in (C and D) show activation kinetics of the outward K⁺ currents (in pA, open symbols) compared to the total charge entered through LTCCs during the prepulse (Q_Ca in pC, closed symbols). (E) Summary data of the half-activation time compared between 50 µM EGTA and 50 µM BAPTA as Ca²⁺ chelators (*P < 0.05).

Figure 4

Activation of SK channels after depletion of the SR Ca²⁺ store. Activation of SK currents by Ca²⁺ influx through LTCCs in the presence of caffeine (A), ryanodine (B) and thapsigargin (C). The pipette solution contained 50 µM BAPTA. The insets in (A–C) show activation kinetics of the outward K⁺ currents (in pA, open symbols) compared to the total charge entered through LTCCs during the prepulse (Q_Ca in pC, closed symbols). (D) Summary data for the half-activation time under different conditions.

Figure 5

Spatial coupling of SK2 and RyR2 channels. (A) STED images of SK2 and RyR2 expression in rabbit ventricular myocytes. Unfiltered and filtered STED images at three Z planes (at the cell surface, 0.18 μm, and 0.36 μm from the cell surface) of SK2, RyR2, and merged images of the SK2 and RyR2. (B) Histograms depicting the frequency and density plots of NND for SK2 and RyR2 (NND(SK2-SK2) and NND(RyR2-RyR2)). (C) Histograms depicting the frequency and density plots of NND(SK2-RyR2) and NND(RyR2-SK2).

Figure 6

Spatial coupling of SK2 and Ca_v1.2 channels. (A) STED images of SK2 and Ca_v1.2 expression in rabbit ventricular myocytes. Unfiltered and filtered STED images of the three Z planes (as in Fig. 5) of SK2 channels, Ca_v1.2, and merged images of the SK2 and Ca_v1.2. (B) Histograms depicting the frequency and density plots of NND for SK2 and Ca_v1.2 (NND(SK2-SK2) and NND(Ca_v1.2-Ca_v1.2). (C) Histograms depicting the frequency and density plots of NND(SK2- Ca_v1.2) and NND(Ca_v1.2-SK2).

Figure 7

Visualization of the spatial distribution of SK2, RyR2, and Ca_v1.2 (AU: arbitrary units).