Ogielska EM and Aldrich RW (1998), A mutation in S6 of Shaker potassium channels d...

XB-ART-14506

J Gen Physiol 1998 Aug 01;1122:243-57. doi: 10.1085/jgp.112.2.243.

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A mutation in S6 of Shaker potassium channels decreases the K+ affinity of an ion binding site revealing ion-ion interactions in the pore.

Ogielska EM , Aldrich RW .

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Under physiological conditions, potassium channels are extraordinarily selective for potassium over other ions. However, in the absence of potassium, certain potassium channels can conduct sodium. Sodium flux is blocked by the addition of low concentrations of potassium. Potassium affinity, and therefore the ability to block sodium current, varies among potassium channel subtypes (Korn, S.J., and S.R. Ikeda. 1995. Science. 269:410-412; Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539-550). The Shaker potassium channel conducts sodium poorly in the presence of very low (micromolar) potassium due to its high potassium affinity (Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539-550; Ogielska, E.M., and R. W. Aldrich. 1997. Biophys. J. 72:A233 [Abstr.]). We show that changing a single residue in S6, A463C, decreases the apparent internal potassium affinity of the Shaker channel pore from the micromolar to the millimolar range, as determined from the ability of potassium to block the sodium currents. Independent evidence that A463C decreases the apparent affinity of a binding site in the pore comes from a study of barium block of potassium currents. The A463C mutation decreases the internal barium affinity of the channel, as expected if barium blocks current by binding to a potassium site in the pore. The decrease in the apparent potassium affinity in A463C channels allows further study of possible ion interactions in the pore. Our results indicate that sodium and potassium can occupy the pore simultaneously and that multiple occupancy results in interactions between ions in the channel pore.

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Species referenced: Xenopus laevis
Genes referenced: kcnb1

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	Figure 2. The S460G:A463C:T469V mutant conducts large sodium currents in the absence of potassium. (A) Sequence comparison between the pore and the S6 region of the Kv2.1 and the Shaker channel. The amino acid differences between the two channels are highlighted. In this study, the starred residues in the S6 of Shaker were mutated to their counterparts in Kv2.1. (B) Current sweeps are from outside-out patches and were elicited by 76-ms voltage ramps from −100 to +200 mV. The pipette solution contained 140 mM NaCl and 0 KCl. With external 140 mM KCl (#1), a large inward potassium current is observed. Replacement of external K with 140 mM NMGCl (#2) allows for large outward sodium currents. The outward current is entirely blocked by 80 nM Agitoxin (#3). (C) Current traces are from an outside-out patch in symmetrical 140 mM NaCl. The currents were elicited by a step from −100 to +100 mV and a repolarization back to −100 mV. Both outward and inward currents are readily blocked by 140 mM external TEA.
	Figure 3. A463C is sufficient for large sodium currents to be observed in the absence of potassium. In all panels, the current sweeps are from outside-out patches and were elicited by voltage ramps from −100 to +200 mV. The pipette solution contained 140 mM NaCl, 0 KCl in all experiments. #1 in all three panels is the current elicited in the presence of 140 mM external KCl. #2 is the current elicited in the presence of 140 mM external NMGCl. S460G (A) and T469V (C) do not readily conduct sodium in the absence of potassium. A463C (B), however, readily conducts sodium in the absence of potassium. The observed sodium current is blocked by 140 mM external TEA (#3).
	Figure 6. In Na+ solutions, the S460G:A463C:T469V mutant has both a shallower steady state voltage dependence and slower deactivation kinetics as compared with K+ solutions. (A) The currents were elicited by 50-ms steps from −100 to +60 mV followed by a 25-ms step to −150 mV. (C) Tail current families were generated by stepping to +150 mV for 20 ms, followed by 20-ms steps from +140 to −200 mV in 10-mV decrements. The families in both panels were recorded in symmetrical 140 mM NaCl. (B) Conductance–voltage relationships in K+ (♦) and Na+ (▴). The K+ data points (n = 12) were best fit with a fourth power Boltzmann with V1/2 = −54.5 mV and z = 2.0 e. The Na+ data points (n = 3) were best fit with V1/2 = −67.4 mV and z = 1.2 e. (D) The time constant of deactivation changes e-fold per 24.1 mV when the permeating ions are K+ (♦; n = 16). Deactivation is slower and the time constant of deactivation changes e-fold per 21.4 mV when the permeating ions are Na+ (▴, n = 9).
	Figure 4. S460G:A463C:T469V and A463C are both selective for K+ in physiological conditions. Representative current traces from S460G:A463C:T469V (A) and A463C (B) elicited by a depolarizing step from a holding potential of −100 to +50 mV and followed by hyperpolarizing steps ranging from −30 to −160 mV in 10-mV decrements. The external solution (A and B) contained (mM): 140 NaCl, 2 KCl, 4 MgCl2, 2 CaCl2, 5 HEPES, pH 7.1. The internal solution contained (mM): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. (C) Instantaneous current–voltage relationship as measured isochronally from the tail current family shown in A. The predicted Nernst reversal potential for K+ under these ionic conditions is −110 mV. The measured reversal potential is −112 ± 4 mV (n = 4). (D) Instantaneous current–voltage relationship as measured from the current family shown in B. The predicted Nernst reversal potential for K+ under these ionic conditions is −110 mV. The measured reversal potential is −106 ± 9 mV (n = 4).
	Figure 5. The gating of S460G:A463C:T469V and A463C in symmetrical potassium solutions. All experiments were performed in symmetrical 140 mM KCl. Representative activation current families for S460G:A463C:T469V (A) and A463C (B). The currents were elicited by 50-ms steps from −100 to +60 mV, followed by a 25-ms step to −65 (A) and −75 (B) mV. (C) Conductance–voltage relationships for ShBΔ6-46 (♦), S460G:A463C:T469V (•), and A463C (▴) were calculated from tail currents such as those in A and B. Error bars represent SEM. The smooth curves through the data represent fourth power Boltzmann fits. The ShBΔ6-46 data points (n = 10) were best fit with V1/2 = −75.7 mV and z = 2.4 e. The triple mutant data (n = 5) were best fit with V1/2 = −60.2 mV and z = 1.8 e. The A463C data (n = 7) were best fit with V1/2 = −61.7 mV and z = 1.1 e. The steady state GV relationships for S460G and T469V were best fitted with V1/2 = −71.5 mV (n = 8) and −76.5 mV (n = 3), respectively (data not shown). Deactivation was studied by tail current families generated by stepping to +50 mV for 25 ms, followed by 25-ms steps from +50 to −200 mV in 10-mV decrements for S460G:A463C:T469V (D) and 15-ms repolarizing steps for A463C (E). Deactivation time constants were obtained from single exponential fits to tail currents such as those shown in D and E. The wild-type (ShBΔ6-46) (♦; n = 7) time constant of deactivation changes e-fold per 30.0 mV, the triple mutant (•, n = 7) changes e-fold per 27.9 mV, and A463C (▴; n = 9) time constant of deactivation changes e-fold per 32 mV. The time constants of deactivation changes for S460G and T469V were e-fold per 31.7 (n = 9) and 28.9 (n = 5) mV (data not shown).
	Figure 7. A463C decreases the apparent potassium affinity of the channel. Data were obtained from inside-out patches in symmetrical 140 mM NaCl. Currents were elicited by voltage steps to +100 mV followed by a step to −100 mV (A) or by voltage ramps from −100 to +200 mV (B). Internally applied potassium blocked the observed currents in a concentration-dependent manner. The data were quantified by measuring percent current block at +100 mV in either steps or ramps versus internal K+ concentration (•) or percent current block at −100 mV in step data (○). (C) The error bars represent SEM. A fit to the data with a single binding isotherm yields an approximate Kd of 6 mM.
	Figure 8. The potassium affinity is influenced by ions residing at neighboring sites. (A) Representative internal K block experiments in the presence of 140 mM symmetrical NaCl are taken from Fig. 7 A and are shown for comparison. (B) Currents elicited by an identical experimental protocol except in the presence of 140 mM external NMGCl and 140 mM internal NaCl. The current was elicited by a voltage step to +100 mV, followed by a step to −100 mV in both cases. (C) The data are quantified as described in Fig. 7 C except that in the external NMGCl experiments only data elicited by voltage steps were used. (•) Blocking data from 140 mM symmetrical NaCl, as shown in Fig. 7 C. (○) Blocking data from experiments with 140 mM external NMGCl and 140 mM internal NaCl. The fits are single binding isotherms yielding values of 2.5 mM for the external NMGCl data and 6.0 mM for the external 140 mM NaCl data.
	Figure 9. A463C decreases the internal Ba++ affinity of the channel. Internal Ba++ affinity was measured in both the wild-type channel and A463C with steps to +100 mV in the presence of symmetrical 140 mM KCl. Wild-type channel block in the presence of 100 (A) and 1,000 (C) μM internal Ba++. A463C channel block in the presence of 100 (B) and 1,000 (D) μM internal Ba++. The data are quantified as percent current versus Ba++ concentration (E). (•) Wild-type data, (○) A463C data. The single binding isotherm fits yield Kds of 75 μM for the wild-type channel and 350 μM for A463C.

References [+] :

Adelman, Blocking of the squid axon potassium channel by external caesium ions. 1978, Pubmed