Low-affinity spermine block mediating outward currents through Kir2.1 and Kir2.2 inward rectifier potassium channels

Abstract

The outward component of the strong inward rectifier K(+) current (I(Kir)) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward I(Kir) and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current-voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 microm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current-voltage relations showed one peak, like those of native I(Kir), outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K(+)] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward I(Kir). When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in two states having different susceptibilities to internal cationic blockers.

Figure 1

SPM block of outward currents through Kir2.2 channels A, representative Kir2.2 currents obtained from an inside-out patch in the absence and presence of the indicated concentrations of SPM. a, currents elicited by test pulses to between −60 and +80 mV in 10 mV increments following a hyperpolarizing prepulse (−40 mV, 10 ms) to relieve the channels from block. The holding potential was 0 mV. b, enlarged outward currents at +20 mV and +70 mV. B, steady-state I–V relations constructed from the data in A. Currents were measured ∼200 ms after the pulse onset; except in the presence of 0.1 μm SPM they were measured ∼600 ms after onset. C, comparison of I–V relations of outward Kir2.2 (a) and Kir2.1 (b) currents in the presence of the indicated concentrations of SPM. Current amplitudes were normalized with respect to the inward-current amplitude at −40 mV in each relation. Shown are mean values for Kir2.2 obtained with 5–6 patches and for Kir2.1 obtained with 8–15 patches. Error bars are not shown when smaller than the symbol.

Figure 7

SPM block of Kir2.1(D172N) channels A, representative currents recorded from an inside-out patch in the absence and presence of the indicated concentrations of SPM. Currents were elicited by test pulses to between −60 and +80 mV in 10 mV increments following a hyperpolarizing prepulse to −40 mV. The holding potential was 0 mV. B, steady-state I–V relations constructed from the currents in A. The right panel shows the reconstructed I–V relations for wild-type Kir2.1 in the presence of 0.1, 1 and 10 μm SPM (see the legend to Fig. 3 for details of the calculation). Red lines depict the components mediated by the ‘low-affinity conductance’. C, G–V relations in the presence of the indicated concentrations of SPM. Mean values from 3 to 4 patches are shown. Error bars are not shown when smaller than the symbol. Continuous lines are the Boltzmann relations fitted using a RT/z₁F value of 8.4 mV. Reconstructed G/G_max values for the wild-type Kir2.1 with only low-affinity block (φ= 0) were superimposed for comparison (red dotted lines); see the legend to Fig. 3 for details of the calculation. D, dissociation constants for the SPM block of Kir2.1(D172N) inferred from the half-blocking voltages of fitted Boltzmann relations (symbols). Fitting to eqn (2) using a RT/z₁F value of 8.4 mV (continuous line) gave a K_d(0) value of 33 μm. K_d(V) values for the SPM block of the wild-type Kir2.1 (black dotted line, high; red dotted line, low) are also shown for comparison.

Figure 3

Comparison of the outward Kir2.1 and Kir2.2 currents based on the two-mode model of SPM block A, K_d(V) values for the high-affinity (black lines) and low-affinity (red lines) SPM block of Kir2.1 (dotted lines) and Kir2.2 (continuous lines). Values were reconstructed using eqn (2). For Kir2.1, K_d(0) values were 0.7 μm (high) and 40 μm (low), and RT/z_iF values were 4.8 mV (high) and 9.1 mV (low) (Ishihara & Ehara, 2004). The range of estimated concentrations of intracellular free SPM in the cardiac ventricle (5–10 μm; Ishihara & Ehara, 2004; Yan & Ishihara, 2005) is denoted by the grey area. B, reconstructed I–V relations for the outward currents in the presence of 0.1, 1 or 10 μm SPM. Current amplitudes were calculated as the product of G/G_max and the membrane potential and were normalized to the value at −40 mV. G/G_max values were calculated using the equation: where K_d1(V) and K_d2(V) are for the high- and low-affinity block, respectively, and φ is the fractional conductance susceptible to the high-affinity block (0.9 for Kir2.1 and 0.935 for Kir2.2). Compare these relations with the experimental results in Fig. 1C. C, reconstructed G–V relations in the presence of 10 μm SPM. Meanings of lines are the same as in A. D, current components mediated by the conductance susceptible to low-affinity block in the presence of 5 or 10 μm SPM (red dotted lines).

Figure 8

SPM block of Kir2.1(E224G) channels A, representative currents recorded from an inside-out patch in the absence and presence of the indicated concentrations of SPM. a, currents elicited by test pulses to between −60 and +80 mV in 10 mV increments from a holding potential of −40 mV b, outward currents at +20, +40 and +60 mV in the presence of 1 μm (left) or 10 μm (right) SPM shown on a longer time scale. B, steady-state I–V relations constructed from the currents in A. Currents were measured at the end of 15, 5, 2 and 1 s test pulses for 1, 10, 100 and 500 μm SPM, respectively, and were normalized to the inward-current amplitude at −60 mV in each SPM concentration. C, G–V relations in the presence of the indicated concentrations of SPM before (a) and after (b) correcting for the instantaneous rectification observed in SPM-free solution. Open circles shown in a denote the instantaneous inward rectification in the SPM-free solution. Mean values from 3 to 4 patches are shown. Error bars are shown in a when larger than the symbol. Continuous lines shown in b are fits to eqn (1); dashed and dotted lines denote the major and minor Boltzmann components, respectively. Fitted RT/z₁F and RT/z₂F values were 7.9 mV and 18 mV, respectively. D, dissociation constants for the SPM block of Kir2.1(E224G) inferred from the half-activation voltages of the fitted Boltzmann components (open circles, high affinity; filled circles, low affinity). Continuous lines are the fits to eqn (2) using the RT/z_iF values of 7.9 mV (high) and 18 mV (low). Fittings gave K_d(0) values of 1.7 μm and 14.9 mm for the high-affinity and low-affinity block, respectively. K_d(V) values for the SPM block of the wild-type Kir2.1 (black dotted line, high; red dotted line, low) are also shown for comparison.

Figure 9

SPM block of Kir2.1(D172N & E224G) channels A, representative currents recorded from an inside-out patch in the presence of the indicated concentrations of SPM. Currents were elicited by test pulses to between −60 and +80 mV in 10 mV increments following a hyperpolarizing prepulse to −40 mV. The holding potential was 0 mV. Note that the current scales vary for the different SPM concentrations. B, steady-state I–V relations constructed from the currents in A. Current amplitudes were normalized to the inward-current amplitude at −60 mV at each SPM concentration. C, G–V relations in the presence of the indicated concentrations of SPM before (a) and after (b) correcting for the instantaneous rectification observed in SPM-free solution. Open circles shown in a denote the instantaneous inward rectification in the SPM-free solution. Mean values from 3 to 4 patches are shown. Error bars are shown in a when larger than the symbol. In b, reconstructed G/G_max values for the Kir2.1(E224G) with only the low-affinity block (φ= 0) were superimposed for comparison (green line, 100 μm; blue line, 500 μm).

Figure 4

SPM block of the outward Kir2.1 currents at low external [K⁺] A, representative currents recorded from inside-out patches in the absence and presence of the indicated concentrations of SPM in 150 mm (a), 50 mm (b) and 20 mm (c) external [K⁺]. The holding potential was set close to V_rev, and short (3 or 20 ms) hyperpolarizing prepulses (∼40 mV more negative than V_rev) were given before applying test pulses. Test pulses were from ∼50 mV more negative than V_rev to ∼80 mV more positive than V_rev in 10 mV increments. Currents in the same [K⁺] were obtained from one patch. B, enlarged outward currents at ∼20 mV and ∼70 mV more positive than V_rev in 150 mm (a), 50 mm (b) and 20 mm (c) external [K⁺]. C, steady-state I–V relations constructed from the data in A: upper panels, I–Vs for the outward currents in the presence of 0.1, 1 or 5 μm SPM; lower panels, I–Vs for the inward and outward currents in the presence of 0.1 or 10 μm SPM.

Figure 6

SPM block of the outward Kir2.1 currents at 5.4 mm external [K⁺] A, representative currents from an inside-out patch with 1, 5 or 10 μm SPM. The holding potential was set at −70 mV (near V_rev), and a 5 ms hyperpolarizing prepulse to −110 mV (∼40 mV more negative than V_rev) was given before applying test pulses from −130 mV to 20 mV in 10 mV increments. Lower panels show enlarged outward currents at −50 mV and 0 mV (∼20 mV and ∼70 mV more positive than V_rev, respectively). NMDG was included in the 5.4 mm K⁺ pipette solution to correct the osmolarity. B, normalized I–V relations for the outward currents. Currents obtained from the same patch with different concentrations of SPM were normalized to the largest amplitude of the outward currents observed at ∼−50 mV in the presence of 5 μm SPM. Mean values from 4 patches (0.1, 1 and 5 μm SPM) and 3 patches (10 μm SPM) are shown. Error bars are not shown when smaller than the symbol.

Figure 2

Voltage dependence of the SPM block of Kir2.2 channels A, G–V relations in the presence of the indicated concentrations of SPM. Mean values from 5 to 6 patches are shown. Error bars are not shown when smaller than the symbol. Continuous lines are fits to eqn (1). B, fitting of G–V relations with the sum of two Boltzmann relations shown on a semilogarithmic scale. Symbols are the mean values shown in A. Continuous lines are fits to eqn (1); dashed and dotted lines show the major and minor Boltzmann components, respectively. RT/z₁F and RT/z₂F values were 4 mV and 9.5 mV, respectively, and the fractional amplitude of the major Boltzmann component (φ) was 0.935. C, dissociation constants for the high-affinity (open squares) and low-affinity (filled squares) block inferred from the half-blocking voltages of the major and minor Boltzmann components, respectively. SPM concentrations were plotted against the half-activation voltages. Straight lines are fits with eqn (2) using the RT/z₁F and RT/z₂F values of the Boltzmann components.

Figure 5

Voltage dependence of the SPM block of Kir2.1 channels at low external [K⁺] A, G–V relations in the presence of the indicated concentrations of SPM. Experiments in which current rundown was negligible were selected for analysis, and G_max at 0.1 μm SPM was used to calculate G/G_max values. Mean values were obtained from 5, 6–8 and 2 patches for 150 mm, 50 mm and 20 mm[K⁺], respectively. For 150 mm and 50 mm[K⁺], error bars are not shown when smaller than the symbol. Statistical errors were not calculated for 20 mm[K⁺]. Vertical lines denote mean values for V_rev (2.0 ± 0.4, –24.9 ± 0.5 and −46.9 mV at 150, 50 and 20 mm K⁺, respectively). Continuous lines are fits to eqn (1): the reductions in the conductances for the inward currents related to the negative shift in the inward I–V relation (Fig. 4C, lower panels) were ignored in the fittings, as before (Ishihara & Ehara, 2004). B, fitting of G–V relations using the sum of two Boltzmann relations shown on a semilogarithmic scale. Symbols are the mean values in A. Continuous lines are fits to eqn (1); dotted lines depict minor Boltzmann components. C, dissociation constants for the high-affinity (open symbols) and low-affinity (filled symbols) block inferred from the fitted Boltzmann components plotted against V – V_rev. Straight lines are the fits to eqn (2) using the RT/z₁F and RT/z₂F values from the fitted Boltzmann relations. D, relations between the fitted K_d(0) values and the external [K⁺] shown on a log–log scale. Straight lines are the theoretical relation: K_d(0) =K_d(0)*exp(z_iln([K⁺]_o/150)), where K_d(0)* is the K_d(0) value at 150 mm[K⁺]_o, and z_i values are 5.5 (high) and 2.9 (low).

Figure 10

Two-mode model for the mechanism of the high-affinity and low-affinity polyamine block of Kir2 channels We propose that Kir2 channels exist in two conformational states with differing susceptibilities to internal cationic blockers. In the ‘high-affinity state’, the block of K⁺ permeation by polyamines occurs within the transmembrane pore. Negatively charged D172 within the transmembrane pore may form the polyamine binding site for the block. An intermediate binding step of polyamines to negatively charged E224 and E299 within the cytoplasmic pore may facilitate this block and its relief (Kubo & Murata, 2001; Xie et al. 2003) and may also cause the ‘instantaneous shallow block’ preceding the time-dependent (steady state) steep block described by Lopatin et al. (1995). In the ‘low-affinity state’, blockade of K⁺ permeation occurs within the cytoplasmic pore, where E224 and E299 together form the polyamine binding site. Polyamines may not access the transmembrane pore due to steric hindrance. The block of Kir2.1(E224G) channel in the ‘high-affinity state’ occurs extremely slowly, and that in the ‘low-affinity state’ is greatly attenuated, due to reduced electrostatic interaction at position 224. The ratio of the low/high affinity state in the presence of internal SPM (1/9) differs from that in the presence of SPD (1/3), and may be regulated by the binding of polyamines to an internal regulatory site (Ishihara & Ehara, 2004).