Cardiac sarcolemmal ATP-sensitive K⁺ (K_ATP) channels form as octamers of four Kir6.2 channel subunits and four SUR2A subunits (Aguilar-Bryan et al. 1998). We have shown previously that certain C-terminal fragments of SUR2A co-immunoprecipitate with Kir6.2, and act as dominant negative polypeptides in HEK 293 cells stably expressing Kir6.2 and SUR2A, reducing the number of functional K_ATP channels (Rainbow et al. 2002). Here, we describe the effects of SUR2A fragments in rat ventricular myocytes.

Ventricular myocytes were isolated enzymatically from humanely killed adult rats, transfected using Lipofectamine 2000 with SUR2A fragments in the pIRES2-EGFP-F vector, and cultured at 37 °C in minimal Eagle’s medium. After 48 h myocytes were transferred to Tyrode solution. Transfected cells were identified by green fluorescence, and studied using patch clamp or video microscopy. The channel opener pinacidil was used to activate K_ATP current.

In cells transfected with vector alone, the current density at 0 mV in 200 µM pinacidil was 7.04 ± 1.22 pA pF^-1 (mean ± S.E.M., n = 6). Transfection with the entire C-terminus of SUR2A (residues 1254-1545) reduced this to 0.39 ± 0.02 pA pF^-1 (n = 6, P < 0.001, ANOVA plus Student-Neumann-Keuls). The 65 amino-acid fragment 1294-1358 was also effective at reducing current density (0.94 ± 0.07 pA pF^-1, n = 6, P < 0.001 versus vector alone). In contrast, no reduction in whole-cell K_ATP current was observed when the distal C-terminus (residues 1358-1545) was expressed (current density 6.30 ± 0.85 pA pF^-1, n = 6), and we have used this fragment as a control. We also investigated K_ATP channel opening in cell-attached patches on myocytes exposed to metabolic inhibition (MI, substrate-free Tyrode, 2 mM NaCN, 1 mM iodoacetate). SUR2A1294-1358 did not affect the time to opening, but reduced the maximum number of channels seen in the patch to about 20 % of that seen in control cells. Transfection with SUR2A1294-1358 delayed action potential failure in cells exposed to MI, and increased the time to contractile failure, but decreased the time to rigor contraction from a control value 4.91 ± 0.14 min (n = 30) to 3.07 ± 0.15 (n = 46, P < 0.001).

Our results suggest that C-terminal fragments of SUR2A containing residues 1294-1358 reduce functional K_ATP channel density in cardiac myocytes, and that this can delay action potential failure and contractile failure but accelerate rigor during MI.

This work was supported by the British Heart Foundation and the MRC.