A phenylalanine at the extracellular side of Kir1.1 facilitates potassium permeation.

The Kir1.1 (ROMK) family of weak inward rectifiers controls K secretion in the renal CCT and K recycling in the renal TALH. A single point mutant of the inward rectifier, F127V-Kir1.

LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b.

Gating of the mammalian inward rectifier Kir1.1 at the helix bundle crossing (HBC) by intracellular pH is believed to be mediated by conformational changes in the C-terminal domain (CTD). However, the exact motion of the CTD during Kir gating remains controversial.

Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA.

The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined.

Residues at the outer mouth of Kir1.1 determine K-dependent gating.

Three residues (E132, F127, and R128) at the outer mouth of Kir1.1b directly affected inward rectifier gating by external K, independent of pH gating. Each of the individual mutations E132Q, F127V, F127D, and R128Y changed the normal K dependence of macroscopic conductance from hyperbolic (Km = 6 ± 2 mM) to linear, up to 500 mM, without changing the hyperbolic K dependence of single-channel conductance.

Modulation of Kir1.1 inactivation by extracellular Ca and Mg.

Kir1.1 inactivation, associated with transient internal acidification, is strongly dependent on external K, Ca, and Mg. Here, we show that in 1 mM K, a 15 min internal acidification (pH 6.

A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity.

ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity.

An intersubunit salt bridge near the selectivity filter stabilizes the active state of Kir1.1.

ROMK (Kir1.1) potassium channels are closed by internal acidification with a pKa of 6.7 +/- 0.

External K activation of Kir1.1 depends on the pH gate.

The inward rectifier Kir1.1 (ROMK) family is gated by both internal pH and external K, where the putative pH gate is formed by the convergence of leucine side chains, near the inner helical bundle crossing at L160-Kir1.1.

Moving the pH gate of the Kir1.1 inward rectifier channel.

Both structural and functional studies suggest that pH gating of the inward rectifier potassium (K) channel, Kir1.1 (ROMK), is mediated by the convergence of four hydrophobic leucines (one from each subunit) near the cytoplasmic bundle-crossing of the inner transmembrane helices. We tested this hypothesis by moving the putative leucine gate from L160-Kir1.

Role of conserved glycines in pH gating of Kir1.1 (ROMK).

Gating of inward rectifier Kir1.1 potassium channels by internal pH is believed to occur when large hydrophobic leucines, on each of the four subunits, obstruct the permeation path at the cytoplasmic end of the inner transmembrane helices (TM2). In this study, we examined whether closure of the channel at this point involves bending of the inner helix at one or both of two highly conserved glycine residues (corresponding to G134 and G143 in KirBac1.

Structural locus of the pH gate in the Kir1.1 inward rectifier channel.

The closed-state crystal structure of prokaryotic inward rectifier, KirBac1.1, has implicated four inner helical phenylalanines near the cytoplasmic side as a possible locus of the channel gate. In the present study, we investigate whether this structural feature corresponds to the physiological pH gate of the renal inward rectifier, Kir1.