Yang L and Palmer LG (2018), Determinants of selective ion permeation in the...

XB-ART-56369

J Gen Physiol 2018 Oct 01;15010:1397-1407. doi: 10.1085/jgp.201812164.

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Determinants of selective ion permeation in the epithelial Na+ channel.

Yang L , Palmer LG .

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The epithelial Na+ channel (ENaC) is a key transporter mediating and controlling Na+ reabsorption in many tight epithelia. A very high selectivity for Na+ over other cations, including K+, is a hallmark of this channel. This selectivity greatly exceeds that of the closely related acid-sensing channels (ASICs). Here, we assess the roles of two regions of the ENaC transmembrane pore in the determination of cation selectivity. Mutations of conserved amino acids with acidic side chains near the cytoplasmic end of the pore diminish macroscopic currents but do not decrease the selectivity of the channel for Na+ versus K+ In the WT channel, voltage-dependent block of Na+ currents by K+ or guanidinium+, neither of which have detectable conductance, suggests that these ions permeate only ∼20% of the transmembrane electric field. According to markers of the electric field determined by Zn2+ block of cysteine residues, the site of K+ block appears to be nearer to the extracellular end of the pore, close to a putative selectivity filter identified using site-directed mutations. To test whether differences in this part of the channel account for selectivity differences between ENaC and ASIC, we substitute amino acids in the three ENaC subunits with those present in the ASIC homotrimer. In this construct, Li:Na selectivity is altered from that of WT ENaC, but the high Na:K selectivity is maintained. We conclude that a different part of the pore may constitute the selectivity filter in the highly selective ENaC than in the less-selective ASIC channel.

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Species referenced: Xenopus laevis
Genes referenced: asic1 gnas

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	Figure 1. Alignment of the C-terminal transmembrane domains of human ASIC1a and rat ENaC subunits. The putative GAS selectivity region, named for the amino-acid sequence in ASIC1, is marked in blue, the conserved acidic amino acids in red, and the sites of Zn2+-sensitive cysteine locations in yellow. Bottom: The transmembrane segments in one subunit of the putative open structure of chicken ASIC (Baconguis et al., 2014; PDB accession no. 4NTW), and a cartoon representation of the transmembrane pore. The relevant elements in TM2 are indicated with the same color scheme.
	Figure 2. Current-voltage relationships in ENaC-expressing oocytes. (A) Currents in the presence of 110 mM extracellular Na+. (B) Currents in the presence of 110 mM extracellular K+. (C) I-V plots show currents as a function of voltage in the presence of Na+ with and without 10 µM amiloride, and the difference currents (INa). (D) Amiloride-sensitive currents in the presence of Na+ or K+.
	Figure 3. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with mutations in the E18′ positions. (A) WT. (B) αE595Q/β/γ. (C) α/βE537Q/γ. (D) α/β/γE549Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+ (I0). Data represent means ± SEM for four to seven oocytes.
	Figure 4. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with mutations in the E21′ positions. (A) WT. (B) αE598Q/β/γ. (C) α/βE540Q/γ. (D) α/β/γE552Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+ (I0). Data represent means ± SEM for three to seven oocytes.
	Figure 5. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with truncations in the β subunit. (A) α/βΤ/γ. (B) αE595Q/βΤ/γE549Q. (C) αE598Q/βΤ/γE552Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+. Data represent means ± SEM for three to six oocytes.
	Figure 6. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with truncations in the β subunit and αC506S mutations. (A) WT. (B) αC506S/βΤ/γ. (C) αC506S E595Q/βΤ/γ. (D) αC506S/βΤE537Q/γE549Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+. Data represent means ± SEM for three to six oocytes.
	Figure 7. Single-channel currents in E18′Q mutants. Currents were recorded in the cell-attached mode at a pipette potential of +100 mV with 110 mM LiCl in the pipette. (A) WT ENaC. (B) αE598Q/β/γ. (C) α/β/γE552Q. (D) Single-channel i-V relationships for the channels shown in A through C. Data represent means ± SEM for two to seven patches. Slopes estimated by linear regression gave conductances of 6.9 ± 0.6 pS (WT), 7.6 ± 0.2 pS (αE598Q/β/γ), and 8.9 ± 0.2 pS α/β/γE552Q. The last value is significantly greater than that of the WT channel (P = 0.04).
	Figure 8. Voltage dependence of block by K+ in oocytes expressing ENaC. (A) INa-V relationships in 20 mM NaCl with 90 mM NMDG+ and no K+, or 90 mM K+. (B) Plot of ln(I0/IK – 1) versus voltage where I0 and IK are currents in the absence and presence of K+, respectively. The slope of the relationship obtained by linear regression gives KK(0) = 450 ± 70 mM, z•δ = 0.20 ± 0.03 (n = 5).
	Figure 9. Voltage dependence of block by guanidinium in oocytes expressing ENaC. (A) INa-V relationships in 30 mM NaCl with 80 mM NMDG+ and no guanidinium or 80 mM guanidinium. (B) Plot of ln(I0/Igdm – 1) versus voltage where I0 and Igdm are currents in the absence and presence of guanidinium, respectively. The slope of the relationship obtained by linear regression gives Kgdm (0) = 320 ± 40 mM, z•δ = 0.24 ± 0.02 (n = 6).
	Figure 10. Effect of Na+ on block by K+ in oocytes expressing ENaC. (A) INa-V relationships in 10 mM Na+ with and without 70 mM K+. (B) INa-V relationships in 40 mM Na+ with and without 70 mM K+. (C) KK at Vm = −150 mV with 10 and 40 mM Na+. Values are significantly different (P = 0.002). (D) KK and KNa as a function of voltage.
	Figure 11. Voltage dependence of block by Zn2+ in oocytes expressing αS583C/β/γ ENaC. (A) INa-V relationships for WT ENaC in 110 mM NaCl with no Zn2+ and 0.2 mM Zn2+. (B) INa-V relationships for ENaC αS583C/β/γ in 110 mM NaCl with no Zn2+ and 0.2 mM Zn2+. (C) Plot of ln(I0/IZn – 1) versus voltage, where I0 and IZn are currents in the absence and presence of Zn2+, respectively, for αS583C/β/γ. The slope of the relationship obtained by linear regression gives KZn(0) = 230 ± 40 µM, z•δ = 0.14 ± 0.01 (n = 5), assuming no stimulation of the channel by Zn2+ (red). Analysis assuming the same activation of WT and αS583C/β/γ gives KZn(0) = 150 ± 20 µM, z•δ = 0.06 ± 0.01 (black).
	Figure 12. Voltage dependence of block by amiloride in oocytes expressing WT and αS583C ENaC. (A) INa-V relationships for WT in 20 mM NaCl with no amiloride or 0.1 µM amiloride (amil). (B) Plot of ln(I0/Iamil – 1) versus voltage where I0 and Iamil are currents in the absence and presence of amiloride, respectively. The slope of the relationship obtained by linear regression gives Kamil = 64 ± 11 nM, z•δ = 0.03 ± 0.01 (n = 6). (C) INa-V relationships for αS583C in 110 mM NaCl with no amiloride or 10−6 M amiloride. (D) Plot of ln(I0/Iamil – 1) versus voltage. The slope of the relationship obtained by linear regression gives Kamil = 2.0 ± 0.2 µM, z•δ = 0.12 ± 0.01 (n = 4).
	Figure 13. Current-voltage relationships in WT ENaC and ENaC(GAS)3. (A and B) Currents were measured in the presence of 110 mM Na+, 110 mM K+, 110 mM guanidinium+, and 110 mM Li+ in oocytes expressing ENaC (A) and ENaC(GAS)3 (B). All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+. Data represent means ± SEM for three to eight oocytes.
	Figure 14. Single-channel currents in WT ENaC and ENaC(GAS)3. Currents were recorded in the cell-attached mode at a pipette potential of +100 mV with 110 mM NaCl or 110 mM LiCl in the pipette. Top: Typical traces from WT and ENaC(GAS)3. Bottom: i-V relationships. Data represent means ± SEM for three to five patches. Slope conductances for WT were 5.3 pS for Na+ and 6.9 pS for Li+. For ENaC(GAS)3, they were 1.6 pS for Na+ and 8.1 pS for Li+.

References [+] :

Anantharam, Open probability of the epithelial sodium channel is regulated by intracellular sodium. 2006, Pubmed, Xenbase