ATP-sensitive potassium (K_ATP) channels are widely distributed in the body and are activated according to the metabolic state of tissues. An important determinant of the activation state of these channels is the degree of phosphorylation. In smooth muscle, K_ATP channels are activated when phosphorylated by protein kinase A (PKA) and deactivated when these residues are dephosphorylated by unknown phosphatases (Clapp et al., 1998). Recently we showed that calcineurin, a calcium-dependent phosphatase which is also known to be functionally coupled to the RII subunit of PKA (Santana et al., 2002), plays a role in the regulation of vascular K_ATP channel function (Wilson et al., 2000). We therefore investigated calcineurin regulation of the putative smooth muscle clone, Kir6.1/SUR2B and whether PKA was involved in this channel regulation. Whole cell currents were recorded in a symmetrical potassium (140 mM) gradient in HEK293 cells stably expressing Kir6.1/SUR2B. The magnitude of currents generated through the K_ATP channel was assessed by their sensitivity to 10 μM glibenclamide. Cells were dialysed with solution containing different free calcium concentrations (0, 18 and 36 nM). Currents were recorded following 10-15 minutes dialysis in the absence or presence of constitutively active calcineurin Aa (CnAα, 100 nM) or β (CnAβ,100 nM), or calcineurin inhibitors, cyclosporin A (CsA, 10 μM) and calcineurin auto-inhibitory peptide (CAP, 100 μM ). Modulation via PKA was investigated using the inhibitors, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 2HCl (H-89, 10 μM) and Rp-2′-O-Monobutyryl-cAMPS (Rp-cAMPS, 100 μM). The magnitude of glibenclamide-sensitive current (I_glib) recorded in Kir6.1/SUR2B cells decreased with increase in the intracellular free calcium concentration; I_glib recorded at -80 mV in 0 nM free Ca²⁺ was 93.9 ± 14.6 pA/pF (n=20), in 18 nM free Ca²⁺ was 61.3± 8.4 pA/pF (n=43) and at 36 nM free Ca²⁺ was 26.2± 9.5 pA/pF (n=11) confirming a role for calcium in the channel regulation. The constitutively active CnAα but not CnAβ significantly (P<0.05, n=6, t-test) attenuated I_glib in the absence of intracellular Ca²⁺. In addition, CAP increased I_glib by 2 fold (P<0.01, n=8, t-test) compared with control cells (n=43). Maximum activation of the channels occurred 10-15 min following dialysis of CAP. On the other hand, the PKA inhibitors H-89 (n=8) and Rp-cAMPS (n=5) significantly (P <0.05, t-test) decreased I_glib in Kir6.1/SUR2B and abolished the increase in current observed with CAP. In summary, our results suggest that CNAα but not CNAβ regulates Kir6.1/SUR2B and that this is achieved by opposing PKA-dependent activation of the channel.