Mikheil Nanazashvili

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 +/- 0.01 in 100 mM external K and a pKa of 7.0 +/- 0.01 in 1 mM external K. Internal acidification in 1 mM K (but not 100 mM K) not only closed the pH gate but also inactivated Kir1.1, such that realkalization did not restore channel activity until high K was returned to the bath. We identified a new putative intersubunit salt bridge (R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter that affected the K sensitivity of the inactivation process. Mutation of either R128-Kir1.1b or E132-Kir1.1b caused inactivation in both 1 mM and 100 mM external K during oocyte acidification. However, 300 mM external K (but not 200 mM Na + 100 mM K) protected both E132Q and R128Y from inactivation. External application of a modified honey-bee toxin, tertiapin Q (TPNQ), also protected Kir1.1 from inactivation in 1 mM K and protected E132Q and R128Y from inactivation in 100 mM K, which suggests that TPNQ binding to the outer mouth of the channel stabilizes the active state. Pretreatment of Kir1.1 with external Ba prevented Kir1.1 inactivation, similar to pretreatment with TPNQ. In addition, mutations that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or stabilized a selectivity filter to helix-pore linkage (V121T-Kir1.1b) also protected both E132Q and R128Y from inactivation in 1 mM K and 100 mM K. Our results are consistent with Kir inactivation arising from conformational changes near the selectivity filter, analogous to C-type inactivation.

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. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased the macroscopic conductance ratio, GRb/GK by 17 ± 3 fold and altered the selectivity sequence from NH4 > K > Tl > Rb >> Cs in wt-Kir1.1 to: Rb > Cs > Tl > NH4 >> K in R128Y, without significant change in the high K/Na permeability ratio of Kir1.1. R128M produced similar, but smaller, increases in Rb, Tl, NH4 and Cs conductance relative to K. R128Y remained susceptible to block by both external Ba and the honeybee toxin, TPNQ, although R128Y had a reduced affinity for TPNQ, relative to wild-type. The effect of R128Y-Kir1.1b on the GRb/GK ratio can be partly explained by a larger single-channel Rb conductance (12.4 ± 0.5 pS) than K conductance (< 1.5 pS ) in this mutant. The kinetics of R128Y gating at -120 mV with Rb as the permeant ion were similar to those of wt-Kir1.1 conducting Rb, but with a longer open time (129 ms vs. 6 ms for wt) and two closed states (13 ms, 905 ms), resulting in an open probability (Po) of 0.5, compared to a Po of 0.9 for wt-Kir1.1, which had a single closed state of 1 ms at -120mV. Single-channel R128Y rectification was eliminated in excised, inside-out patches by removal of internal Mg and polyamines. The large increase in the Rb/K conductance ratio, with no change in K/Na permeability or rectification, is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter.

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 4 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 the L160-Kir1.1b to other positions along the inner transmembrane helix, and measuring inward current and conductance as functions of internal pH, using the Xenopus oocyte heterologous expression system. Results of these studies indicated that it was possible to replace the putative inward rectifier pH gate at L160-Kir1.1b by either a leucine or methionine at 157-Kir1.1b (G157L-L160G or G157M-L160G). Although both leucine and methionine gated the channel at 157-Kir1.1b, residues of similar hydrophobicity (tyrosine and valine) did not. Hence, hydrophobicity was a necessary but not a sufficient condition for steric gating at 157. This was in contrast to the 160-Kir1.1b locus, where side-chain hydrophobicity was both a necessary and sufficient property for steric gating. Homology models were constructed for all mutants that expressed significant whole-cell currents, using the closed-state coordinates of the prokaryotic inward rectifier, KirBac1.1. Models of mutants that retained pH gating were too narrow at the bundle crossing to permit hydrated K ion permeation in the closed-state. On the other hand, mutants that lost pH gating had ample space at the bundle crossing for hydrated K permeation in the closed-state. These results support our hypothesis that hydrophobic leucines at the cytoplasmic end of the inner transmembrane helices comprise the principal pH gate of Kir1.1, a gate that can be relocated from 160-Kir1.1b to 157-Kir1.1b.

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. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.

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. Crystal structures and single-molecule fluorescence resonance energy transfer of KirBac channels have implied a rigid body rotation and/or a contraction of the CTD as possible triggers for opening of the HBC gate. In our study, we used lanthanide-based resonance energy transfer on single-Cys dimeric constructs of the mammalian renal inward rectifier, Kir1.1b, incorporated into anionic liposomes plus PIP2, to determine unambiguous, state-dependent distances between paired Cys residues on diagonally opposite subunits. Functionality and pH dependence of our proteoliposome channels were verified in separate electrophysiological experiments. The lanthanide-based resonance energy transfer distances measured in closed (pH 6) and open (pH 8) conditions indicated neither expansion nor contraction of the CTD during gating, whereas the HBC gate widened by 8.8 ± 4 Å, from 6.3 ± 2 to 15.1 ± 6 Å, during opening. These results are consistent with a Kir gating model in which rigid body rotation of the large CTD around the permeation axis is correlated with opening of the HBC hydrophobic gate, allowing permeation of a 7 Å hydrated K ion.

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. This suggests that E132, F127, and R128 are responsible for maximal Kir1.1b activation by external K. In addition, these same residues were also essential for recovery of Kir1.1b activity after complete removal of external K by 18-Crown-6 polyether. In contrast, charge-altering mutations at neighboring residues (E92A, E104A, D97V, or Q133E) near the outer mouth of the channel did not affect Kir1.1b recovery after chelation of external K. The collective role of E132, R128, and F127 in preventing Kir1.1b inactivation by either cytoplasmic acidification or external K removal implies that pH inactivation and the external K sensor share a common mechanism, whereby E132, R128, and F127 stabilize the Kir1.1b selectivity filter gate in an open conformation, allowing rapid recovery of channel activity after a period of external K depletion.