ATP-modulated K+ channels play an important role in regulating membrane excitability during metabolic stress. To characterize such K+ channels from the human brain, single channel currents were studied in excised inside-out patches from freshly dissociated human neocortical neurons. Three currents that were sensitive to physiological concentrations of ATP and selectively permeable to K+ were identified. One of these currents had a unitary conductance of approximately 47 pS and showed a strong inward rectification with symmetric K+ concentrations across the membrane. This K+ current was inhibited by ATP in a concentration-dependent manner with an IC50 (half-inhibition of channel activity) of approximately 130 microM. Channel activity also was suppressed by ADP, non-hydrolyzable ATP analogue AMP-PNP, and sulfonylurea receptor/ channel blocker glibenclamide. The second K+ current had a unitary conductance of approximately 200 pS and showed a weak inward rectification. Similarly, this current was inhibited by ATP (IC50 = 350 microM), AMP-PNP, and glibenclamide. Unlike the small-conductance ATP-inhibitable K+ channel (S-KATP), activation of this large-conductance K+ channel (L-KATP) required the presence of micromolar concentration of Ca2+ in the internal solution, but charybdotoxin did not inhibit this channel. The third K+ current was also Ca2+ dependent and had a large conductance (approximately 280 pS). It was inhibited by external charybdotoxin, iberiotoxin, and tetraethylammonium. In contrast to the other two KATP channels, ATP enhanced channel open-state probability and unitary conductance, and glibenclamide at concentration of 10-20 microM had no inhibitory effect on this current. K+ channels that have single-channel and pharmacological properties similar to these three human ATP-modulated K+ channels also were observed in experiments on rat neocortical neurons. These results therefore indicate that KATP channels are expressed in human neocortical neurons, and two distinct KATP channels (S-KATP and L-KATP) exist in the human and rat neurons. The observation that ATP at different concentrations modulates different K+ channels suggests that metabolic rate may be continuously sensed in neurons with resulting alterations in neuronal membrane excitability.