Sabirov RZ et al. (1997), A conserved arginine residue in the pore region...

XB-ART-15645

J Gen Physiol 1997 Dec 01;1106:665-77.

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A conserved arginine residue in the pore region of an inward rectifier K channel (IRK1) as an external barrier for cationic blockers.

Sabirov RZ , Tominaga T , Miwa A , Okada Y , Oiki S .

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The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg148, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2. 1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K+ > Rb+ > NH4+ >> Na+) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg2+ and Ca2+, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg2+ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs+ and Ba2+, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg2+ or Ca2+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg148 is located between the external blocking site for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in the IRK1 channel. It is concluded that Arg148 serves as a barrier to cationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.

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Species referenced: Xenopus
Genes referenced: kcnj12 kcnj2 pkm

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	Figure 2. Macroscopic currents recorded from Xenopus oocytes injected with mRNA of the wild-type (WT), and mixture of WT and the R148H mutant at different ratios. (A) Representative current traces. Currents were recorded by applying steps of 1,000-ms duration from +40 to â140 mV in 10-mV decrements. Holding potential was â30 mV. (B) Current amplitude at â100 mV as a function of fractional R148H content in mRNA mixture. (C) Representative instantaneous I-V curves of the data in A. (D) The chord conductances normalized at â140 mV as a function of voltage. âª, WT; â¡, fm = 0.25; â, fm = 0.5; âµ, fm = 0.75.
	Figure 10. Cation selectivity of WT and R148H/WT hybrid channels. Currentâvoltage relationships for WT (left) and hybrid (fm = 0.75; right) channels were observed in the presence of 10 mM KCl and 80 mM XCl, where X was either K+, NMDG+, Na+, Rb+, NH4+, or Cs+. The holding potential was â30 mV and currents were evoked by steps from +40 to â140 mV in 5-mV increments.
	Figure 3. Single-channel currents of WT and R148H/WT (fm = 0.5) hybrid channels. (A) Representative single-channel currents recorded from Xenopus oocytes injected with mRNA of WT (left) and the mixture of WT and the R148H mutant (fm = 0.5; middle and right). Representative traces from channels of two different conductances are shown from coinjected oocytes. Downward and upward arrows indicate the beginning and the end of the voltage step (500 ms), respectively, from the holding potential of â30 mV. (B) Single-channel I-V curves for WT (âª) and hybrid (â and âµ) channels. (C) Distribution of single-channel currents for WT (top) and coinjected R148H/WT (fm = 0.5) channels (bottom) at â100 mV obtained from different patches and oocytes (n = 21 and 60 for WT and hybrid channels, respectively).
	Figure 4. Sensitivity to external Mg2+ of WT and R148H/WT hybrid channels. (A) Representative traces of macroscopic currents recorded from Xenopus oocytes injected with WT (left) and a mixture of WT and R148H mutant mRNAs at fm = 0.5 (right). The scale bar indicates 20 Î¼A for the left traces and 6 Î¼A for the right. Pulse protocol was the same as that of Fig. 2 A while the duration was 500 ms. (B) Instantaneous I-V curves at different concentrations of Mg2+ for WT and hybrid channels (fm = 0.5). (C) Concentration dependency of the Mg2+ blockage of the fractional current at â140 mV. Curves represent fitting to Eq. 1 with Kd = 4.38 Â± 0.19 mM (WT; âª), 3.21 Â± 0.19 mM (fm = 0.25; â¡), 1.92 Â± 0.14 mM (fm = 0.5; â), and 1.31 Â± 0.06 mM (fm = 0.75; âµ). The unblocked fraction of currents was 0.31 Â± 0.07 for WT, 0.20 Â± 0.10 for fm = 0.25, 0.17 Â± 0.01 for fm = 0.5, and 0.05 Â± 0.01 for fm = 0.75. (D) Representative traces of single-channel currents for WT (left) and hybrid (fm = 0.5; right) channels recorded with patch pipettes filled with the bath solution containing 5 mM Mg2+. Downward and upward arrows indicate the beginning and the end of the voltage step, respectively, applied repetitively to â100 mV from a holding potential of â30 mV.
	Figure 5. Voltage dependence of Mg2+ block conferred by the mutation R148H. (A) Chord conductances normalized to â140 mV for WT in 25 mM Mg2+ (âª), fm = 0.5 in 25 mM Mg2+ (â), fm = 0.75 in 20 mM Mg2+ (âµ). Similar results were obtained in the presence of 5, 10, and 15 mM Mg2+. (B) Voltage dependency of the apparent Kd of Mg2+ block for WT and hybrid channels. Symbols are the same as in Fig. 2. Lines represent fitting to Eq. 2 with Î´ plotted in C. Kd(0) = 4.3 mM (wild type), Kd(0) = 4.5 mM (fâm = 0.25), Kd(0) = 3.6 mM (fâm = 0.5), and Kd(0) = 3.8 mM (fâm = 0.75). (C) Electrical distances for Mg2+ blocking as a function of the mutant ratio in mRNA mixtures.
	Figure 6. pH sensitivity conferred by the mutation, R148H. (A) pH dependency of the steady state current amplitude at âˆ’140 mV normalized to the control current (IpH/IpH = 8.5; â–ª). The line fitted to the hybrid channel data represents the titration curve of a single group with pK = 7.23 Â± 0.03. [Mg2+]bath = 1 mM. (B) Steady state single-channel current traces, pH 7.06, recorded at âˆ’100 mV. The WT channel (30 pS) does not show sublevels. Long-lasting sublevels are shown for the 25 pS channel (middle) and flickering sublevels are shown for the 20 pS channel (right). Transition from the sublevel to the closed level was observed (for example, B, middle second trace). (C) The effect of pH on Mg2+ block of WT (â–ª) and hybrid (fm = 0.5; â—‹) channels. The degree of current block by 5 mM Mg2+ is plotted as a function of pH. Here, Io is the current at âˆ’140 mV in Mg2+-free solution and I is the current in the presence of 5 mM Mg2+. The line fitted to the hybrid channel data represents the titration curve of a single group with a pK = 7.06 Â± 0.03.
	Figure 7. Ca2+ blockade of WT and hybrid channels coexpressed at different fm. (A) Doseâresponse relationships. Curves represent fitting to Eq. 1 with Kd = 9.4 Â± 2.7 mM (WT; âª), Kd = 6.4 Â± 1.2 mM (fm = 0.25; â¡), Kd = 5.5 Â± 1.0 mM (fm = 0.5; â), and Kd = 3.9 Â± 0.6 mM (fm = 0.75; âµ). (B) Chord conductances normalized at â140 mV as a function of voltage for WT and 60 mM Ca2+ (âª), fm = 0.5 and 60 mM Ca2+ (â), and fm = 0.75 and 60 mM Ca2+ (âµ). Essentially similar results were obtained in three experiments. (C) Voltage dependency of dissociation constants for Ca2+ block of WT and hybrid channels. Symbols are the same as those in Fig. 2. Lines represent fitting to Eq. 2 with Î´ plotted in D. (D) Electrical distances for block by Ca2+ as a function of mutant content in mRNA mixtures.
	Figure 8. Blocking rate of Cs+ and Ba2+ in WT and R148H/WT hybrid channels. (A) Representative current traces showing the time course of Cs+ and Ba2+ block of WT (top) and hybrid (âfm = 0.5; bottom) channels. (B) Single-exponential fitting of blocking time course for Cs+ (left) and Ba2+ (right) at â140 mV. Dotted lines correspond to fitting with time constants of 0.82 and 0.37 ms for WT and fm = 0.5, respectively, for Cs+ block and 149 and 57 ms for Ba2+ block. (C) Voltage dependence of the time constant for Cs+ (left) and Ba2+ (right) blocking for WT (âª) and hybrid (fm = 0.5; â) channels. Lines are fits to Eq. 3. For Cs+ block, k(0) = 164 Â± 32 sâ1, Î´ = 0.35 Â± 0.04 for WT and k(0) = 166 Â± 90 sâ1, Î´ = 0.49 Â± 0.11 for the hybrid. For Ba2+ block, k(0) = 0.13 Â± 0.01 sâ1, Î´ = 0.37 Â± 0.01 for WT and k(0) = 0.22 Â± 0.002 sâ1, Î´ = 0.39 Â± 0.001 for the hybrid. The ÎÎGâ¡ (=ÎGâ¡hybrid â ÎGâ¡WT) values were â0.02 RT for Cs+ block and â0.47 RT for Ba2+ block.
	Figure 9. Cs+ blocking of WT and hybrid channels. (A) Representative I-V relationships for WT (â¢) and hybrid (fm = 0.5; â) channels at different concentrations of Cs+. Currents for hybrid channels were scaled up to match the WT current at â100 mV in the absence of blocker. (B) Fractional currents at â140 mV as a function of Cs+ concentration. The curve represents fitting of the WT data to Eq. 1 with Kd = 13.7 Â± 0.4 Î¼M. 14.6 Â± 0.26 Î¼M for fm = 0.25, 15.03 Â± 0.30 Î¼M for fm = 0.5 and 17.24 Â± 0.91 Î¼M for fm = 0.75.(C) Voltage dependence of dissociation constants. A line represents fitting of WT data to Eq. 2 with Kd (0 mV) = 5.62 Â± 0.75 mM and Î´ = 1.09 Â± 0.03. Symbols are as in Fig. 2.

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