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Figure 2. Monoamino alkanes cause weak inward rectification of Kir2.1 currents. (left) Kir2.1 currents recorded from an inside-out membrane patch in response to steps to voltages between −80 and +70 mV after a prepulse to −80 mV from a holding potential of 0 mV as indicated. In each case, the control recordings were obtained after removal of rectification after patch excision, and the patch was then exposed to 100 μM monoamines as indicated. Scale bars: 2 nA and 5 ms. (right) Steady state current–voltage relationships plotted from the above current traces.
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Figure 3. Longer monoamines cause inward rectification with very slow unblock rates. (left) Kir2.1 currents recorded from an inside-out membrane patch in response to steps to voltages between −120 and +60 mV after a prepulse to −120 mV from a holding potential of 0 mV as indicated. In each case, the control recordings were obtained after removal of rectification after patch excision, and the patch was then exposed to 100 μM monoamines as indicated. Scale bars: 1 nA and 5 ms. (right) Steady state current–voltage relationships plotted from the above current traces.
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Figure 4. Monoamine blocking affinity increases with alkyl chain length. (A) Relative conductance (GRelative, see text)–voltage relationships obtained for block of Kir2.1 currents by different alkyl chain length monoamines at a concentration of 100 μM (mean values from n = 2–6 patches, ±SEM where n > 2). Smooth lines are fits of a Boltzmann function (see text) to averaged data. (B) V1/2 of block as a function of alkyl chain length (N) from fits to the data in A. (C) The effective valence of block (Z) as a function of alkyl chain length (N) from fits to the averaged data in A.
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Figure 5. Monoamine block kinetics are voltage dependent. Time constant of block (τBlock) obtained from single exponential fits to currents like those shown in Figs. 2 and 3 plotted versus membrane potential (Vm).
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Figure 6. Monoamine unblock kinetics are strongly dependent on alkyl chain length. (A) Kir2.1 currents recorded from an inside-out membrane patch in response to voltage steps to potentials between 0 and −150 mV after a prepulse to +50 mV, from a holding potential of 0 mV as indicated. In each case, the control recordings were obtained after removal of rectification after patch excision, and the patch was then exposed to 100 μM monoamines as indicated. Scale bars: 2 nA and 5 ms. (B) Time constant of unblock (τOff) obtained from exponential fits (see text) to currents like those shown in A plotted versus membrane potential (Vm).
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Figure 7. Diamino alkanes cause inward rectification of Kir2.1 with fast kinetics. (left) Kir2.1 currents recorded from inside-out membrane patches in response to steps to voltages between −100 and +60 mV (A), or between −80 and +70 or +80 mV (B) after a prepulse to −100 (A) or −80 (B) mV from a holding potential of 0 mV. In each case, the control recordings were obtained after removal of rectification after patch excision, and the patch was then exposed to 100 μM diamines as indicated. Scale bars: 2 nA and 5 ms. (right) Steady state current–voltage relationships plotted from the above current traces.
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Figure 8. Diamino alkane blocking affinity and effective valency increase with chain length. (A) Relative conductance (GRelative, see text)–voltage relationships obtained for block of Kir2.1 currents by different alkyl chain length diamines at a concentration of 100 μM. Smooth lines are best fits of a Boltzmann function (see text) to representative experiments in Fig. 7. (B) V1/2 of block as a function of alkyl chain length (N) from fits to the data in A. (C) The effective valence of block (Z) as a function of alkyl chain length (N) from fits to the data in A. In B and C, X indicates the V1/2 and Z obtained for block by 100 μM spermidine.
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Figure 9. (A) Diamine block and unblock kinetics are much faster than monoamine kinetics. Time constants (τ) of block and unblock by 100 mM MA8 and DA8 obtained from experiments shown in Figs. 3, 6, 7, and 10. Plotted versus voltage. The line fitted to the MA8 data corresponds to a single site-blocking model with kon,0mV = 5 × 10−4 ms−1 · μM−1 and koff,0mV = 0.03 ms−1, Zon = 2.2, Zoff = 1.6. The line corresponding to DA8 is the same model with kon and koff 100× faster. The horizontal dashed line indicates the approximate limits of resolution for these experiments, so that block kinetics for 100 μM DA8 were too rapid to measure (plotted below the line). Asterisks indicate τ for block by 1 μM DA8 (see text). (B) Representative current traces show that increasing the alkyl chain length slows channel block. Traces show block by 1 μM DA8, DA10, and DA12 at 50 mV after a prepulse to −80 mV. The capacitance transient has been digitally subtracted from the depolarizing pulses, and all traces have been scaled to the amplitude of current at −80 mV. Dashed line indicates zero current level. (C) The valence of diamine block is independent of blocker concentration. Effective valence of block (Z) by DA10 as a function of blocker concentration, obtained from a single patch subjected to a voltage protocol like that shown in Fig. 7.
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Figure 10. Diamine unblock kinetics are dependent on alkyl chain length. (A) Kir2.1 currents recorded from an inside-out membrane patch in response to voltage steps to voltages between 0 and −100 or −150 mV after a prepulse to +50 mV, from a holding potential of 0 mV as indicated. The currents were obtained sequentially (DA6, Control, DA8, DA10, DA9, DA12) with exposure to 100 μM diamines as indicated. Scale bars are 1 nA and 2 or 5 ms as indicated. (B) Time constant of unblock (τOff) obtained from exponential fits (see text) to currents in A, plotted versus membrane potential (Vm).
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Figure 11. Block by phenyl amines is considerably weaker than block by alkylamines. (A) Kir2.1 currents recorded from an inside-out membrane patch in response to voltage steps to voltages between −80 and +70 mV after a prepulse to −80 mV from a holding potential of 0 mV. Currents are shown in the presence of 10 μM DA7, 100 μM PhMA, 100 μM PhEA, or 1 mM pPhDA. In each case, the post-control currents were obtained after washing out the test solution. Scale bars: 5 nA for pPhDA, PhEA, and DA7, 2 nA for PhMA, and 5 ms. (B) Steady state GRelative–voltage relationships for the currents shown in A. The continuous lines are fits to the Boltzmann equation (see text).
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Figure 12. Accommodation of polyamines within the pore of Kir channels. Kir pores are hypothesized to have a long (>20 Å) inner vestibule with strong electronegativity provided by the ring of negative charges at the “rectification controller” position (D172). While more peripheral negative charges (e.g., E224) may provide additional peripheral amine binding sites, a single diamine (bottom) can occupy the deep pore, the two amines being equidistant from the center of the ring of negativity at the rectification controller. As alkyl chain length increases, the head amine is pushed deeper into the pore, displacing K+ ions to the outside of the cell. Monoamines (top) can also occupy the pore, but the head amine remains in the ring of electronegativity provided by the rectification controller, the alkyl chain stretching back out of the pore.
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