Spassova M and Lu Z (1999), Tuning the voltage dependence of tetraethylammo...

XB-ART-12417

J Gen Physiol 1999 Sep 01;1143:415-26.

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Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel.

Spassova M , Lu Z .

Abstract
To understand the role of permeating ions in determining blocking ion-induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.

PubMed ID: 10469731
PMC ID: PMC2229458

Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: kcnj1

Article Images: [+] show captions

	Figure 2. Tetraethylammonium inhibition curves. The fractions of unblocked currents (I/Io) were plotted against the concentration of intracellular TEA ([TEAint]) for several representative membrane voltages. Curves superimposed on the data correspond to least-squares fits using I/I0 = TEAKobs/(TEAKobs + [TEAint]), where TEAKobs is the observed TEA equilibrium dissociation constant. The alkali metal ions (100 mM) contained in the pipette solution were as indicated.
	Figure 1. Voltage-dependent block of the ROMK1 channel by intracellular TEA in various extracellular alkali ions. Macroscopic I–V curves of the ROMK1 channel were recorded in the absence or presence of various concentrations of intracellular TEA. The pipette solutions contained 100 mM of either K+, Rb+, Cs+, Na+, or NMG+. The bath solution contained 100 mM K+ in all cases.
	Figure 5. Dependence of TEAKobs(0 mV) on the concentration of extracellular alkali metal ions. TEAKobs(0 mV) values (mean ± SEM, n = 5) are plotted against the concentration of extracellular K+, Rb+, Cs+, or Na+. The values of TEAKobs(0 mV) were obtained as shown in Fig. 3. (A–C) When the concentration of K+, Rb+, or Cs+ was reduced, Na+ was used as a substitute. (D) The Na+-free solution contained 100 mM NMG+. The lines superimposed on the data represent fits of (see discussion).
	Figure 3. Dependence of TEAKobs on membrane voltage. The natural logarithms of TEAKobs values obtained in Fig. 2 were plotted against membrane voltage. The lines superimposed on the data are least-squares fits of the Woodhull equation (Woodhull 1973), lnTEAKobs = lnTEAKobs(0 mV) − TEA(zδ)obsFVm/RT. The alkali metal ions (100 mM) contained in the pipette solution were as indicated.
	Figure 4. The values of TEAKobs(0 mV) and TEA(zδ)obs determined in various extracellular alkali metal ions. TEAKobs(0 mV) and TEA(zδ)obs values (mean ± SEM, n = 5) obtained in the presence of 100 mM of each of the four alkali metal ions are presented in A and B, respectively. The data were obtained as shown in Fig. 3.
	Figure 7. Effects of intracellular permeant ions on channel blockade by intracellular TEA. (A and B) TEAKobs(0 mV) (mean ± SEM, n = 5) are plotted against the concentration of extracellular K+ in A and Rb+ in B. Data in the open symbols were obtained in 100 mM intracellular K+, while data in the closed symbols were obtained in 100 mM intracellular Rb+. The lines superimposed on the data correspond to . (C and D) The values of TEA(zδ)obs (mean ± SEM, n = 5) are plotted against the concentration of extracellular K+ in C and Rb+ in D. Data in the open symbols were obtained in 100 mM intracellular K+, while data the in closed symbols were obtained in 100 mM intracellular Rb+. The curves superimposed on the data have no theoretic meaning.
	Figure 6. Blockade of the ROMK1 channel by intracellular TEA in the presence of intracellular Rb+. Macroscopic I–V curves of the ROMK1 channel were recorded in the absence or presence of various concentrations of intracellular TEA. The pipette solutions contained 100 mM K+ (A), 100 mM Rb+ (B), 20 mM K+ (C), and 20 mM Rb+ (D). The bath solution contained 100 mM of Rb+ in all four cases.
	Figure 8. A kinetic state diagram. Extracellular ion X and intracellular TEA bind competitively to a channel (Ch).

References [+] :

ARMSTRONG, ANOMALOUS RECTIFICATION IN THE SQUID GIANT AXON INJECTED WITH TETRAETHYLAMMONIUM CHLORIDE. 1965, Pubmed