Mechanisms of Coupling

ROMK channels are regulated by internal pH (pH i ) and extracellular K (K o ). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H , increasing the apparent pKa for inhibition by 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K o , consistent with coupling between the responses to pH i and K o . The ion selectivity sequence of the activation of the channel by cations was K Rb NH 4 Na , similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K o . We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba 2 block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H . We conclude that decreasing pH I increases the sensitivity of ROMK2 channels to K o by altering the properties of the selectivity filter. key words: ROMK2 • IRK1 • renal K secretion • Xenopus oocytes • transmembrane helix I N T R O D U C T I O N ROMK channels from the kidney are regulated by intracellular pH; acidification of the cytoplasm below pH 7.0 results in a sharp decrease in whole-cell conductance and in single-channel open probability through a cooperative process with Hill coefficients greater than two (Tsai et al., 1995; Fakler et al., 1996; Choe et al., 1997; McNicholas et al., 1998; Chanchevalap et al., 2000). This response probably accounts for decreases in renal K secretion known to occur during acidosis (Malnic et al., 2000). A lysine residue near the NH 2 terminus (K61 in ROMK2), presumably within the first transmembrane-spanning domain, is critical for this response, and may be the site whose ionization state controls the channel’s function (Fakler et al., 1996; Choe et al., 1997; Schulte et al., 1999). Other amino acids, both in the NH 2 terminus and the COOH terminus, have been shown to modulate the response by shifting the apparent pKa (Choe et al., 1997; Schulte et al., 1999; Chanchevalap et al., 2000). These channels can also be closed by decreasing the concentration of K in the extracellular fluid (Doi et al., 1996; Sackin et al., 2001). This is a property also shared by some voltage-gated K channels in which slow, “C-type” inactivation is promoted by low extracellular K concentrations (Lopez-Barneo and Aldrich 1993; Liu et al., 1996; Starkus et al., 1997). In ROMK, the effects of extracellular K and intracellular pH are interdependent; decreasing K o makes the channels more sensitive to pH i (Doi et al., 1996; Sackin et al., 2003). Furthermore, mutation of the same critical lysine (K61 of ROMK2) abolishes K o sensitivity (Schulte et al., 2001; Sackin et al., 2003). Interactions between pH and K effects may be explained by conformational changes that propagate from the inside aspect of the channel protein to the outside or vice versa (Schulte et al., 2001). An alternative mechanism has also been proposed in which the effects of external cations are exerted through occuAddress correspondence to Lawrence G. Palmer, Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Fax: (212) 774-7860; email: [email protected] T h e Jo u rn al o f G en er al P h ys io lo g y on Jne 5, 2017 D ow nladed fom Published March 29, 2004