MODIFICATION BY PROTONS OF FROG SKELETAL-MUSCLE K-ATP CHANNELS - EFFECTS ON ION CONDUCTION AND NUCLEOTIDE INHIBITION

1. The molecular mechanisms underlying pH regulation of skeletal muscl e ATP-sensitive K+ (K-ATP) channels were studied using the patch clamp technique in the inside-out configuration. Two effects of intracellul ar protons were studied in detail: the decrease in magnitude of single -channel currents and the increase in open probability (P-0) of nucleo tide-inhibited channels. 2. The pH dependence of inward unit currents under different ionic conditions was in poor agreement with either a d irect block of the pore by protons or an indirect proton-induced confo rmational change, but was compatible with the protonation of surface c harges located near the cytoplasmic entrance of the pore. This latter electrostatic mechanism was modelled using Gouy-Chapman-Stern theory, which predicted the data accurately with a surface charge density of a bout 0.1 negative elementary charges per square nanometre and a pK (pH value for 50% effect) value for protonation of these charges of 6.25. The same mechanism, i.e. neutralization of negative surface charges b y cation binding, could also account for the previously reported reduc tion of inward unit currents by Mg2+. 3. Intracellular alkalization di d not affect P-0 of the K-ATP channels. Acidification increased P-0. I n the presence of 0.1 mM ATP (no Mg2+), the channel activation vs. pH relationship could be fitted with a sigmoid curve with a Hill coeffici ent slightly above 2 and a pK value of 6. This latter value was depend ent on the ATP concentration, decreasing from 6.3 in 30 mu M ATP to 5. 3 in 1 mM ATP. 4. Conversely, the channel inhibition vs. ATP concentra tion curve was shifted to the right when the pH was lowered. At pH 7.1 , the ATP concentration causing half-maximal inhibition was about 10 m u M. At pH 5.4, it was about 400 mu M. The Hill coefficient values rem ained slightly below 2. Similar effects were observed when ADP was use d as the inhibitory nucleotide. 5. These results confirm that a recipr ocal competitive link exists between proton and nucleotide binding sit es. Quantitatively, they are in full agreement with a steady-state mod el of a K-ATP channel possessing four identical protonation sites (mic roscopic pK, 6) allosterically connected to the channel open state and two identical nucleotide sites (microscopic ATP dissociation constant , similar to 30 mu M) connected to the closed state.