Epithelial Na+ channel subunit stoichiometry - PubMed (original) (raw)

Epithelial Na+ channel subunit stoichiometry

Alexander Staruschenko et al. Biophys J. 2005 Jun.

Abstract

Ion channels, including the epithelial Na(+) channel (ENaC), are intrinsic membrane proteins comprised of component subunits. Proper subunit assembly and stoichiometry are essential for normal physiological function of the channel protein. ENaC comprises three subunits, alpha, beta, and gamma, that have common tertiary structures and much amino acid sequence identity. For maximal ENaC activity, each subunit is required. The subunit stoichiometry of functional ENaC within the membrane remains uncertain. We combined a biophysical approach, fluorescence intensity ratio analysis, used to assess relative subunit stoichiometry with total internal reflection fluorescence microscopy, which enables isolation of plasma membrane fluorescence signals, to determine the limiting subunit stoichiometry of ENaC within the plasma membrane. Our results demonstrate that membrane ENaC contains equal numbers of each type of subunit and that at steady state, subunit stoichiometry is fixed. Moreover, we find that when all three ENaC subunits are coexpressed, heteromeric channel formation is favored over homomeric channels. Electrophysiological results testing effects of ENaC subunit dose on channel activity were consistent with total internal reflection fluorescence/fluorescence intensity ratio findings and confirmed preferential formation of heteromeric channels containing equal numbers of each subunit.

PubMed Disclaimer

Figures

FIGURE 1

FIGURE 1

FIR-TIRF controls. (A) Fluorescence micrographs showing the plasma membrane of COS-7 cells expressing eCFP-m and eYFP-m at cDNA ratios of 1:0, 0:1, 1:1, 2:1, 1:2, 3:1, and 1:3 with the top and bottom rows showing eCFP (pseudocolored cyan) and eYFP (pseudocolored yellow) emissions, respectively. Cells were imaged with TIRF microscopy. (B) Unscaled best-fit linear regression lines of data points for eCFP and eYFP emissions from cells expressing eCFP-m and eYFP-m at cDNA ratios of 1:0 and 0:1 (yellow); 1:1 (red); 2:1 and 1:2 (blue); and 3:1 and 1:3 (green). Also shown is the (dashed red) regression line for eCFP and eYFP emissions from cells expressing the CGY-m fusion protein, which contains the fluorophores as a FRET pair. (C) Results from panel B scaled using the constant C. Black dashed lines predict eCFP-m and eYFP-m membrane levels at the indicated expression ratios.

FIGURE 2

FIGURE 2

FRET has little impact on FIR. (Left) Unscaled best-fit linear regressions lines of data points for eCFP and eYFP emissions from cells expressing eCFP-m and eYFP-m at cDNA ratios of 2:1 and 1:2 in the absence of FRET (blue) and with hypothetical FRET of 30% between these fluorophores (red; 2:1′ and 1:2′). Also shown are the FIR values _k_1 and _k_2, and _k_′1 and _k_′2 in the absence and presence of FRET, respectively. (Right) Results from panel A scaled using the constant C (blue) and C′ (red). The green lines represent the maximum possible error introduced by FRET for the 2:1 and 1:2 regression lines. Black dashed lines indicated predicted eCFP-m and eYFP-m membrane levels at the indicated expression ratios.

FIGURE 3

FIGURE 3

ENaC expressed in the membrane. Fluorescence micrographs of COS-7 cells coexpressing eCFP-_α_ENaC + eYFP-_β_ENaC and untagged _γ_ENaC (top row), and eCFP-_β_ENaC + eYFP-_α_ENaC and untagged _γ_ENaC (bottom row) collected with total internal reflection fluorescence microscopy. Fluorescence emissions from eCFP- (pseudocolored green) and eYFP- (pseudocolored red) tagged ENaC within the plasma membrane are shown in the left and middle columns. The right column shows merged images.

FIGURE 4

FIGURE 4

The relative amounts of _α_- and _β_-subunits in membrane ENaC are the same. Scatter plot of eCFP versus eYFP fluorescence emissions collected with TIRF microscopy from cells coexpressing eCFP-_α_ENaC + eYFP-_β_ENaC and untagged _γ_ENaC (A) and eCFP-_β_ENaC + eYFP-_α_ENaC and untagged _γ_ENaC (C). Solid red lines are the best-fit linear regression lines (m = fluorescence intensity ratio for panels A and C, which are _k_1 and _k_2); black dashed lines predict subunit relations at the indicated stoichiometry; red, blue, and green circles represent data points from cells transfected with equal amounts of eCFP- and eYFP-tagged subunit and those with twice and three times as much eCFP- versus eYFP-tagged subunit, respectively. Scatter plots of the quotient of eCFP/eYFP fluorescence emissions from panel A (B) and panel C (D) as a function of the relative eCFP- to eYFP-tagged subunit expression level.

FIGURE 5

FIGURE 5

The relative amounts of _β_- and _γ_-subunits in membrane ENaC are the same. Scatter plot of eCFP versus eYFP fluorescence emissions collected with TIRF microscopy from cells coexpressing eCFP-_β_ENaC + eYFP-_γ_ENaC and untagged _α_ENaC (A) and eCFP-_γ_ENaC + eYFP-_β_ENaC and untagged _α_ENaC (C). Solid red lines are the best-fit linear regression lines (m = fluorescence intensity ratio for panels A and C, which are _k_1 and _k_2); black dashed lines predict subunit relations at the indicated stoichiometry; red, blue, and green circles represent data points from cells transfected with equal amounts of eCFP- and eYFP-tagged subunit and those with twice and three times as much eCFP- versus eYFP-tagged subunit, respectively. Scatter plots of the quotient of eCFP/eYFP fluorescence emissions from panel A (B) and panel C (D) as a function of the relative eCFP- to eYFP-tagged subunit expression level.

FIGURE 6

FIGURE 6

The relative amounts of _α_- and _γ_-subunits in membrane ENaC are the same. Scatter plot of eCFP versus eYFP fluorescence emissions collected with TIRF microscopy from cells coexpressing eCFP-_α_ENaC + eYFP-_γ_ENaC and untagged _β_ENaC (A) and eCFP-_γ_ENaC + eYFP-_α_ENaC and untagged _β_ENaC (C). Solid red lines are the best-fit linear regression lines (m = fluorescence intensity ratio for panels A and C, which are _k_1 and _k_2); black dashed lines predict subunit relations at the indicated stoichiometry; red, blue, and green circles represent data points from cells transfected with equal amounts of eCFP- and eYFP-tagged subunit and those with twice and three times as much eCFP- versus eYFP-tagged subunit, respectively. Scatter plots of the quotient of eCFP/eYFP fluorescence emissions from panel A (B) and panel C (D) as a function of the relative eCFP- to eYFP-tagged subunit expression level.

FIGURE 7

FIGURE 7

Increasing expression eCFP-tagged ENaC subunits increases total cellular eCFP fluorescence emissions. Summary graph of the quotient of eCFP/eYFP fluorescence emissions as a function of the relative eCFP- to eYFP-tagged subunit expression level collected with wide-field epifluorescence microscopy from the cells in Fig. 6 coexpressing eCFP-_α_ENaC + eYFP-_γ_ENaC and untagged _β_ENaC (•) and eCFP-_γ_ENaC + eYFP-_α_ENaC and untagged _β_ENaC (▪).

FIGURE 8

FIGURE 8

ENaC has a relative subunit stoichiometry of 1_α_:1_β_:1_γ_. (A) Overlays of typical macroscopic Na+ currents in CHO cells transfected with 0.6, 0.2, and 0.2 _μ_g (top), and 0.6, 0.6, and 0.6 _μ_g (bottom) of _α_-, _β_-, and _γ_ENaC before and after amiloride (noted by arrow). Currents elicited by voltage ramping from 60 to −100 mV. (B) Summary graph of ENaC activity (amiloride-sensitive current density at −80 mV) in CHO cells transfected with _α_-, _β_-, and _γ_ENaC cDNAs at the noted concentrations. The asterisk (*) is versus all other groups.

FIGURE 9

FIGURE 9

ENaC has a limiting stoichiometry of 1_α_:1_β_:1_γ_. Scatter plots of eCFP versus eYFP fluorescence emissions collected with TIRF microscopy from cells coexpressing (A) eCFP-_α_ENaC + eYFP-_β_ENaC + eYFP-_γ_ENaC (red circles) and eYFP-_α_ENaC + eCFP-_β_ENaC + eCFP-_γ_ENaC (blue circles); (B) eCFP-_β_ENaC + eYFP-_α_ENaC + eYFP-_γ_ENaC (red circles) and eYFP-_β_ENaC + eCFP-_α_ENaC + eCFP-_γ_ENaC (blue circles); and (C) eCFP-_γ_ENaC + eYFP-_α_ENaC + eYFP-_β_ENaC (red circles) and eYFP-_γ_ENaC + eCFP-_α_ENaC + eCFP-_β_ENaC (blue circles). Solid red (_k_1) and blue (_k_2) lines are the best-fit linear regression lines and black dashed lines predict subunit relations at the indicated stoichiometry. For all experiments, ENaC subunit cDNAs were expressed at 0.2 _μ_g each.

Similar articles

Cited by

References

    1. Adachi, M., K. Tachibana, Y. Asakura, S. Abe, J. Nakae, T. Tajima, and K. Fujieda. 2001. Compound heterozygous mutations in the gamma subunit gene of ENaC (1627delG and 1570–1G→A) in one sporadic Japanese patient with a systemic form of pseudohypoaldosteronism type 1. J. Clin. Endocrinol. Metab. 86:9–12. - PubMed
    1. Benos, D. J., and B. A. Stanton. 1999. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J. Physiol. (Lond.). 520:631–644. - PMC - PubMed
    1. Canessa, C. M., J. D. Horisberger, and B. C. Rossier. 1993. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 361:467–470. - PubMed
    1. Canessa, C. M., L. Schild, G. Buell, B. Thorens, I. Gautschi, J. D. Horisberger, and B. C. Rossier. 1994. Amiloride-sensitive epithelial Na channel is made of three homologous subunits. Nature. 367:463–467. - PubMed
    1. Corey, S., G. Krapivinsky, L. Krapivinsky, and D. E. Clapham. 1998. Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh. J. Biol. Chem. 273:5271–5278. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources