Structural Locus of the pH Gate in the Kir1.1 Inward Rectifier Channel

Abstract

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, Kir1.1 (ROMK, KCNJ1). Kir1.1 is endogenous to the mammalian renal collecting duct and the thick ascending limb of Henle and is strongly gated by internal pH in the physiological range. It has four leucines (L160-Kir1.1b), homologous to the phenylalanines of KirBac1.1, which could function as steric gates near the convergence of the inner (M2) helices. Replacing these Leu-160 residues of Kir1.1b by smaller glycines abolished pH gating; however, replacement with alanines, whose side chains are intermediate in size between leucine and glycine, did not eliminate normal pH gating. Furthermore, a double mutant, constructed by adding the I163M-Kir1.1b mutation to the L160G mutation, also lacked normal pH gating, although the I163M mutation by itself enhanced the pH sensitivity of the channel. In addition to size, side-chain hydrophobicity at 160-Kir1.1b was also important for normal pH gating. Mutants with polar side chains (L160S, L160T) did not gate normally and were as insensitive to internal pH as the L160G mutant. Hence, either small or highly polar side chains at 160-Kir1.1b stabilize the open state of the channel. A homology model of the Kir1.1 closed state, based on the crystal structure of KirBac1.1, was consistent with our electrophysiological data and implies that closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines at the cytoplasmic apex of the inner transmembrane helices. In the open state, K crosses the pH gate together with its hydration shell.

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

“…We initially selected to mutate highly conserved residues including two hydrophobic residues at the predicted membrane/cytosol interface and a series of positive and negatively charged residues in the distal portion of S6. Hydrophobic residues located at the narrowest point of the channel where the S6 helices cross over each other are known to play important roles in the gating of other ion channels (18,20,21,37). In a previous study we found that mutation of a candidate highly conserved residue (Phe-2592) had little effect on channel function (29).…”

“…In addition to making direct (or indirect) contacts with the N-terminal ligand-binding region of the channel, it has been speculated that the loop may also interact with the C-tail (14) as observed in several other channels (17,(21)(22)(23)(24)(25). This possibility was experimentally tested using fusion proteins encoding the C-tail and S4S5 loop.…”

The Recent Evolution of a Symbiotic Ion Channel in the Legume Family Altered Ion Conductance and Improved Functionality in Calcium Signaling

“…In these cases, the K + channels are involved in setting the threshold for Ca 2+ spike initiation and in limiting the duration of each spike (Golding et al, 1999). Reference conductance levels for K + channels mediating action potentials in animal cells are, for example, 200 pS for large conductance BK (big potassium) channels (Lancaster and Nicoll, 1987), 10 pS for small conductance SK (small conductance calcium-activated potassium) channels (Köhler et al, 1996), and 30 to 50 pS for intermediate/moderate conductance ROMK (renal outer medullary potassium) channels (Sackin et al, 2005). Thus, during evolution, the Ser-to-Ala substitution in the filter region converted a large conductance channel (Lj-CASTOR and Lj-POLLUX) to a moderate conductance channel (DMI1) with enhanced functionality in symbiosis.…”

Inhibition of MAPK stimulates the Ca 2+ -dependent big-conductance K channels in cortical collecting duct

“…Because of their high open probability and channel density, SK channels (ROMK) are thought to be largely responsible for K secretion in the connecting tubule and CCD under physiological conditions. SK channels are sensitive to cytosolic changes in pH (22,23) and regulated by protein kinase A (24,25), protein kinase C (26), and protein tyrosine kinase. The latter is involved in mediating the effect of K restriction on SK channels (27).…”