Scanning the topography of polyamine blocker binding in an inwardly rectifying potassium channel

Abstract

Steeply voltage-dependent inward rectification of Kir (inwardly rectifying potassium) channels arises from blockade by cytoplasmic polyamines. These polycationic blockers traverse a long (>70 Å) pore, displacing multiple permeant ions, en route to a high affinity binding site that remains loosely defined. We have scanned the effects of cysteine modification at multiple pore-lining positions on the blocking properties of a library of polyamine analogs, demonstrating that the effects of cysteine modification are position- and blocker-dependent. Specifically, introduction of positively charged adducts results in two distinct phenotypes: either disruption of blocker binding or generation of a barrier to blocker migration, in a consistent pattern that depends on both the length of the polyamine blocker and the position of the modified cysteine. These findings reveal important details about the chemical basis and specific location of high affinity polyamine binding.

FIGURE 1.

Dimeric cysteine substituted constructs. A, graphic depiction of the Kir channel inner cavity, with shaded clouds to indicate contrasting models for regions involved in high affinity polyamine block. B, construction of tandem-linked dimers of Kir6.2[C166S][N160D]. Assembled channels have negatively charged aspartates occupying the rectification controller position (N160D) in each subunit. The second subunit (back half, dark blue) includes a substituted cysteine. C, location of cysteine substitutions at pore-lining positions in the Kir inner cavity (numbering corresponds to Kir6.2; equivalent numbering for Kir2.1 is in parentheses).

FIGURE 2.

MTSEA and MTSET modification of Kir6.2 position 164C and other inner cavity residues. A and B, spermine (SPM) block (100 μm) of the Kir6.2[164C] dimer was assessed before and after modification with MTSET or MTSEA, at voltages between −100 and +50 mV. C, expanded traces showing the kinetics of spermine block (−20 mV) and unblock (−50 mV), before and after modification by MTSET (top) or MTSEA (bottom), as indicated. D, conductance-voltage relationships illustrating block by 100 μm spermine in unmodified (Unmod) dimeric L164C channels (n = 12) and after modification with MTSEA (n = 3) or MTSET (n = 9). E–G, parameters describing the effects of modification in 157C, 160C/164D, and 164C channels. Conductance-voltage relationships were fit with Boltzmann equations to determine the V_½ of 100 μm spermine block (F) and the effective valence of block (G). Error bars in panels E–G indicate S.E.

FIGURE 3.

MTSEA and MTSET modification of Kir6.2 position 157C. Inside-out patches expressing the Kir6.2[157C] dimer were pulsed between −100 and +50 mV, in control or 100 μm spermine (SPM). Pulse protocols were repeated after complete modification with either MTSET (A) or MTSEA (B). C, expanded traces illustrating the blocking rate in 100 μm spermine at −20 mV and the unbinding rate at −50 mV, before and after MTSET (top) or MTSEA (bottom) modification, as indicated. D, conductance-voltage relationships illustrating the voltage dependence of block in control (n = 12) and after modification with MTSEA (n = 4) or MTSET (n = 6). Error bars indicate S.E.

FIGURE 4.

MTSEA and MTSET modification of Kir6.2 position 160C. A Kir6.2 dimer was constructed carrying N160D in the front subunit, and N160C/L164D in the back subunit. This manipulation allowed examination of modification at position 160C while maintaining four negatively charged residues in the inner cavity. Steady-state block by 100 μm spermine (SPM) was assessed before and after modification with either MTSET (A) or MTSEA (B), as described in the legend for Fig. 2. C, schematic demonstrating the overall design of the dimeric channel. SH indicates the sulfhydryl group of the cysteine (N160C) available for modification in half of the subunits. D, conductance-voltage relationships illustrating the voltage dependence of block in control (n = 11) and after modification with MTSEA (n = 5) or MTSET (n = 6). Kinetics of spermine block and unblock in the 160C/164D dimer were markedly faster than channels with four N160D residues and were not examined in detail. Error bars indicate S.E.

FIGURE 5.

Blockade of Kir6.2[N160D] channels by ammonium, tetramethylammonium, and spermine. A, currents elicited in Kir6.2[N160D] channels between −100 and +50 mV, in the presence of the indicated blockers. B, conductance-voltage relationships summarizing the experiments presented in A. Data are fit with a single Boltzmann function. C, sample currents elicited from Kir6.2[N160D] channels, illustrating spermine blockade alone or in the presence of 100 mm ammonium (NH₄⁺) or tetramethylammonium (TMA⁺). D, conductance-voltage relationships summarizing experiments in B. Data are fit with a sum of two Boltzmann functions: G(V) = A/(1 + e∧(z^aF(V − V_½^a)/kT) + (1 − A)/(1 + e∧(z^aF(V − V_½^a)/kT) (superscript characters are used to distinguish components, not as exponents). Error bars in panels B and D indicate S.E.

FIGURE 6.

Acceleration of spermine unbinding by internal ammonium. A–C, sample currents elicited from Kir6.2[N160D] channels, illustrating spermine unbinding at voltages between −80 and −10 mV, alone or in the presence of 100 mm ammonium (NH₄⁺) or tetramethylammonium (TMA⁺). D, normalized currents depicting spermine unbinding at −50 mV, in control or in the presence of NH₄⁺ or TMA⁺, as indicated. E, summarized data illustrating the rate of spermine (SPM) unbinding between −40 mV and −80 mV, in the presence of 100 mm NH₄⁺ or TMA⁺. Error bars indicate S.E.

FIGURE 7.

Effects of MTSET modification at 164C are blocker-dependent. A, blockade of the Kir6.2[L164C] dimer was examined in the presence of 10 μm PG-11098, a synthetic spermine analog, before and after modification by MTSET. B, schematic illustrating potential interpretation, with blockers longer than spermine extending closer toward the positive charge introduced at position 164C. SH indicates the sulfhydryl group of the cysteint (L164C) available for modification in half of the subunits. C, conductance-voltage relationships before (n = 7) and after (n = 7) MTSET modification illustrate a rightward shift and smaller effective valence of PG-11098 block after MTSET. Error bars indicate S.E. D, kinetics of PG-11098 unbinding before (left panel) and after (right panel) MTSET modification. Patches were pulsed to +50 mV, in control (gray) or 10 μm PG-11098 (black), and repolarized to −50 mV to observe blocker unbinding. Prepulses to +90 mV were also used to ensure significant channel block by PG-11098 after MTSET modification, although PG-11098 unbinding remained rapid.

FIGURE 8.

Localization of blockers in the Kir6.2 pore. Polyamine blockers are shown in fully extended conformations alongside a structural model of Kir6.2. Blocker dimensions range from “short” (i.e. spermine) to blockers that span almost the entire length of the channel pore (e.g. PG-11179). For each substituted cysteine, the kinetics of blocker unbinding at −50 mV are presented before and after modification with MTSEA (and MTSET where determined). At deep sites (L157C, top row), modification accelerates blocker unbinding. At shallower sites (164C, 169C, 212C), a barrier phenotype emerges in which modification slows the kinetics of blocker unbinding. Blocker/position combinations that exhibit a barrier phenotype are highlighted in red. The barrier phenotype emerges with modification at progressively shallower positions for progressively longer blockers, and the pattern suggests that the trailing end of PG-11179 extends beyond position 169C, but not position 212C.

FIGURE 9.

Kinetic effects of MTSEA modification at Kir6.2 positions 169C and 212C. A, sample data of MTSEA modification of Kir6.2[N160D][C166S][M169C] dimeric channels blocked with PG-11179. Channels were blocked with a +50-mV pulse and then repolarized to −50 mV to observe the PG-11179 unbinding rate from unmodified channels (gray box). In the second step, channels were blocked at +50 mV and then exposed to a solution containing PG-11179 + 100 μm MTSEA and repolarized to −50 mV to observe the blocker unbinding rate from partially modified channels. B and C, expanded data illustrating relevant unbinding kinetics observed in similar experiments using PG-11179 (B) or spermine (C). MTSEA modification of residue 169 slows spermine unbinding, but has no discernible effect on PG-11179 unbinding. Graphics depict spermine and PG-11179 orientations relative to position 169C (indicated by positive charges). Post-mod, after modification; Pre-mod, before modification. D–F, identical experiments were performed with spermine and PG-11179 and modification of position 212C. MTSEA modification of 212C slows unbinding of both spermine and PG-11179.

FIGURE 10.

Blocker- and position-dependent emergence of the barrier phenotype by modification of pore-lining cysteines. For each substituted cysteine, the kinetics of blocker unbinding at −50 mV after modification have been normalized to the unbinding kinetics in unmodified channels. In cases where the unbinding rate after modification cannot be resolved, the τ_control/τ_modified ratio has been arbitrarily assigned a value >1 to indicate the observed acceleration of unbinding. Error bars indicate S.E.

FIGURE 11.

Blocker- and position-dependent effects of modification on blocker potency. A–D, for positions 169C and 212C; extremely slow (60 s) voltage ramps between −80 mV and +60 mV were used to assess the voltage dependence of block for various blockers (100 μm). E, ΔV_½ was determined as the difference in V_½ of block for modified versus unmodified channels (n = 3–7 per condition). V_½ values of block were derived from either voltage step protocols (e.g. Fig. 2) or slow voltage ramps, as depicted in panels A–D. Error bars indicate S.E.

FIGURE 12.

Slow blocker binding in MTSEA-modified 169C and 212C channels. A, exemplar data illustrating the effects of MTSEA modification of Kir6.2[S212C] dimeric channels on blocking kinetics of PG-11098 or spermine (both at 100 μm). Patches were pulsed from −80 mV to +50 mV after the indicated manipulations. Pre-mod, before modification. B, summarized data describing spermine and PG-11098 blocking kinetics in unmodified and MTSEA-modified 169C and 212C channels (n = 3–5 per recording condition). MTSEA modification of these positions profoundly slows blocker binding. Error bars indicate S.E.