Cysteine Accessibilities of Inner Vestibule of ROMK under Open and Low pH Induced Closed State: Evidence Showing That the Locus of pH Gating Lies above the M2 Bundle Crossing

ROMK channels (Kir1.x) are considered to be essential for K + secretion in kidney and thus for the balance of K + in the body. ROMK channels are sensitive to changes of intracellular pH, with acidification favoring channel closure. In order to localize the pH‐dependent gate, we studied the reactivity of methanethiolsulfonate (MTS) reagents with cysteines engineered into an MTS –insensitive template named ROMK1YZ. Accessibility of MTSEA and MTSET to thiol either above or below the M2 bundle crossing was studied in inside‐out macroscopic patches. When the channels were open, both of the reagents irreversibly oxidized a cysteine naturally occurring at position 175 above the bundle crossing, leading to complete block of current. C175 was inaccessible to MTSET under the completely closed state induced by pH 6.0 on the cytoplasmic side of the channel. In contrast, MTSEA, a smaller molecule with the same charge as MTSET, was able to modify the same thiol in the closed as well as the open state. The possibility that MTSEA, which is lipid soluble, might gain access to C175 through the membrane is unlikely as the reagent had no significant effects when added to the extracellular fluid. Similar studies were also performed on the mutant ROMK1YZG223C with an exogenous cysteine residue engineered into the cytoplasmic pore. MTSET but not MTSEA modification completely blocked the channels under open state. The accessibility of C223 to MTSET was not restricted after the channels were closed by low pH. The simplest interpretation is that pH gating involves conformation changes on the M2 segments, but that these changes are insufficient to close the channel. The most important pH gate lies above the M2 bundle crossing, perhaps at the selectivity filter. (Supported by NIH grant DK27847)