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

Abstract

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, G(Rb)/G(K) by 17 ± 3 fold and altered the selectivity sequence from NH(4) > K > Tl > Rb >> Cs in wt-Kir1.1 to: Rb > Cs > Tl > NH(4) >> 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, NH(4) 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 G(Rb)/G(K) 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 -120 mV. Single-channel R128Y rectification was eliminated in excised, insideout patches with symmetrical Rb solutions. 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.

Figure 1

Whole-cell current-voltage relations for wt-Kir1.1b, with either 100 mM extracellular K (black) or 100 mM extracellular Rb (green). Currents were measured on the same oocyte in response to applied clamp voltages between -200 mV and +200 mV. Oocyte internal [K] was approximately 100 mM and acetate buffers were used to clamp the oocyte internal pH at 7.8. Fitted lines indicate either K (black) or Rb (green) inward conductances. Inset shows raw data.

Figure 2

Whole-cell current-voltage relations for R128Y-Kir1.1b, with either 100 mM extracellular K (black) or 100 mM extracellular Rb (green). Currents were measured on the same oocyte in response to applied clamp voltages between -200 mV and +200 mV. Oocyte internal [K] was approximately 100 mM and acetate buffers were used to clamp the oocyte internal pH at 7.8. Fitted lines indicate either K (black) or Rb (green) inward conductances. Magenta curve indicates the average I-V relation for an uninjected oocyte bathed in 100 mM external K. Inset shows raw data.

Figure 3

Whole-cell current-voltage relations for wt-Kir1.1b, with either 100 mM extracellular K (black) or 100 mM extracellular Cs (green). Currents were measured on the same oocyte in response to applied clamp voltages between -200 mV and +200 mV. Oocyte internal [K] was approximately 100 mM and acetate buffers were used to clamp the oocyte internal pH at 7.8. Inward conductances reflect either Cs (green) or K (black) permeation. Outward conductances reflect flow of K out of the oocyte. Inset shows raw data.

Figure 4

Whole-cell current-voltage relations for R128Y-Kir1.1b, with either 100 mM extracellular K (black) or 100 mM extracellular Cs (green). Currents were measured on the same oocyte in response to applied clamp voltages between -200 mV and +200 mV. Oocyte internal [K] was approximately 100 mM and acetate buffers were used to clamp the oocyte internal pH at 7.8. Fitted lines indicate either K (black) or Cs (green) inward conductances. Outward conductances reflect flow of K out of the oocyte. Magenta curve indicates the average I-V relation for an uninjected oocyte bathed in 100 mM external K. Inset shows raw data.

Figure 5

Semi-log plot of R128Y reversal potentials associated with progressive replacement of external Na by: K (open squares), Rb (closed circles), Tl (open triangles) or NH₄ (open circles), at constant ionic strength. Permeability ratios were fit by Eqs 1–3 (Methods) and given in Table 2. Inset shows an example of R128Y inward and outward currents during isosmolar replacement of external Na by Rb.

Figure 6

External K is a competitive inhibitor of inward Rb current in R128Y. Total inward whole-cell conductance was normalized to the maximal conductance at each external [K], which occurred at an external Rb concentration of 200 mM. Curves were drawn from a competitive inhibitor model (Eq 4, Methods). Fitted parameters are given in the text.

Figure 7

Comparison of TPNQ dose-response curves for ROMK vs. R128Y. Binding and block of wt-Kir1.1 (ROMK) by TPNQ at external pH 7, K_D = 1.5 × 10^-9 M (red, open diamonds); and binding and block of R128Y-Kir1.1b by TPNQ at external pH 7, K_D = 1.5 × 10^-7 M (black, closed squares).

Figure 8

Internal acidification in 100 mM Rb solutions reversibly closed R128Y with a pKa of 6.7 ± 0.03 (black). The same acidification in 100 mM external K closed R128Y with a pKa of 6.8 ± 0.03 (red), similar to the pKa of 6.7 ± 0.02 for wt-Kir1.1b. however, R128Y recovered very slowly during realkalization in 100 mM K (dashed red line).

Figure 9

Single-channel inward Rb currents through a cell-attached patch on an R128Y oocyte. The bath solution consisted of: 0 Mg, 2 mM Ca and 10 mM K, which depolarized the oocyte to -45 mV. The patch pipette contained 100 mM Rb, 0 Mg, 2 Ca, and pipette holding potentials were varied between +50 mV and +150 mV relative to the bath (ground). One R128Y channel in the patch, having inward Rb conductance of 14.2 pS. Records were sampled at 5 khz & filtered at 300 Hz.

Figure 10

Single-channel current-voltage relation showing inward Rb current but no discernable outward K current through the R128Y cell-attached patch of Figure 9. Oocyte bathed in 10 mM K, 0 Mg, 2 mM Ca, which depolarized the oocyte to -45 mV. Pipette: 100 RbCl, 0 Mg, 2 Ca, 0.5 mM LaCl₃. Inward single-channel Rb conductance = 14.2 pS.

Figure 11

Histograms for the R128Y inward Rb current of Figure 9 at a V_pch of -120 mV (inside negative). Oocyte was bathed in 10 mM K, 0 Mg, 2 mM Ca, which depolarized the oocyte to -45 mV. Pipette: 100 RbCl, 0 Mg, 2 Ca, 0.5 mM LaCl₃ at a pipette holding potential of +75 mV. (a) Mean open time = 129 ms (1,254 events, one channel). (B) Two closed states with mean closed times = 13 ms (88%) and 905 ms (12%).

Figure 12

Voltage dependence of open probability (Po) for the single-channel Rb current of R128Y in Figure 9, compared to wt-Kir1.1 Rb permeation (dashed line). Curve is the best fit to the Boltzman Eq: Po = Bottom + (Top - Bottom)/(1 + exp((V₅₀ - V)/slope)), where V₅₀ = -117 mV and slope = 30, for R128Y and V₅₀ = -181 mV and slope = 41, for wild-type.

Figure 13

Voltage dependence of mean open time for the single-channel Rb current of R128Y in Figure 9, compared to wt-Kir1.1 Rb permeation (dashed line). The data were fit to a simple exponential: Tau (open time) = α•exp(β•V), where α = 3280 for R128Y and 58 for wt, and β = 0.03 for R128Y and 0.02 for wt.

Figure 14

Voltage dependence of the 2 closed times (squares, circles) for the single-channel R128Y Rb currents in Figure 9, compared to wt-Kir1.1 Rb currents (open triangles, dashed line), which have only a single closed state.

Figure 15

Rb currents from an excised R128Y patch with 100 mM RbCl, zero Mg, 2 mMCa, 0.5 mM LaCl₃ in the pipette and 100 mMRbCl, zero Mg, zero Ca, 4 mM NaF in the bath at pH = 9. Upward deflections from the thick dashed line (closed state) correspond to outward current at V_pch = +100 mV (top trace). Downward deflections from the dashed line denote inward currents at V_pch = -100 mV and -150 mV respectively.

Figure 16

Excised current-voltage relation for Rb currents through R128Y. Single channel Rb conductance was 10.2 pS. There was no obvious rectification of single-channel current in the absence of bath Mg and polyamines. Pipette: 100 mM RbCl, 0 Mg, 0 Ca, 0.5 mM LaCl₃. Bath: 100 mM RbCl, 0 Mg, 0 Ca, 4 NaF, pH 9.

Figure 17

The modified honeybee toxin, TPNQ (cyan), blocks wt-Kir1.1 by binding to the outer mouth of the channel at F129-Kir1.1b (yellow side chains). TPNQ also blocks the mutant channel, R128Y-Kir1.1b, but with a 100 fold higher K_D, consistent with the Tyr side-chain of R128Y (cyan) impeding close approach of the toxin. picture was drawn with PYMOL from our homology model, based on Kir2.2, (see Methods) and the known structure of TpNQ.