To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5'-triphosphate (ATP)-sensitive K+ (KATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal KATP currents (at 0 mV) induced by metabolic inhibition were 48.9+/-2.8 pA/pF in control (n=48), 34.3+/-3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5+/-3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na+ current and inward-rectifying K+ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of KATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional KATP channels. Furthermore, in four out of seven control cells tested, outward K+ currents were stimulated by diazoxide, which is a potent K+ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional KATP channels. The larger population of KATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2.