Counting membrane-embedded KCNE beta-subunits in functioning K+ channel complexes

Abstract

Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting alpha-subunits with specific regulatory beta-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of alpha- and beta-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K(+) channel inhibitor and its iterative application to tally the number of beta-subunits in a KCNQ1/KCNE1 K(+) channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K(+) channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE beta-subunit modulation of voltage-gated K(+) channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K(+) channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE beta-subunits.

Fig. 1.

A cartoon depiction of one modification cycle of the iterative counting strategy used to determine the number of KCNE1 subunits in a KCNQ1 K⁺ channel complex by using a derivatized charybdotoxin (CTX). Each complete round of chemical treatment modifies one E1 subunit, allowing for the direct counting of KCNE subunits in the K⁺ channel complex.

Fig. 2.

Structures of the CTX reagents and the mechanism by which the cleavable linker is severed by reductant. (a) Cysteine-modifying charybdotoxins. (b) Tris(2-carboxyethyl)phosphine (TCEP) cleavage of bis(N-phenylcarbamoyl)disulfane linkers and the resultant products.

Fig. 3.

Characterization of CTX-Clv. (Left) Overlays of the total current elicited by a 20-mV pulse from oocytes expressing Q1/E1 and Q1/E1_T14C complexes. Scale bars are 0.5 μA and 0.5 s. (Right) Normalized CTX-sensitive current (I/I_max). Oocytes were depolarized every 30 s and the data points were obtained at the end of a 5-s pulse. Gray and red data points correspond to the raw traces on the left. (a) Q1/E1_T14C complexes were treated with 10 nM CTX-Clv, followed by washout and 1 mM TCEP treatment, demonstrating one complete modification cycle. (b) Irreversible inhibition of Q1/E1 complex requires an extracellular cysteine in E1 because wild-type Q1/E1 complexes are reversibly inhibited by only 10 nM CTX-Clv. (c) Irreversible inhibition of Q1/E1_T14C by 10 nM CTX-Clv is completely prevented in the presence of excess (500 nM) CTX (unlabeled). (d) A tethered CTX-Clv prevents modification of the unmodified E1 peptides in Q1/E1_T14C complexes by CTX-Mal.

Fig. 4.

KCNQ1 K⁺ channel complexes contain two KCNE1 peptides. (a) Iterative counting of E1_T14C subunits in a Q1 K⁺ channel complex. (Upper) Raw data showing the total current for each round. Scale bar is 0.5 μA and 0.5 s. (Lower) Q1/E1_T14C complexes were treated with two rounds (10 nM CTX-Clv, washout, 1 mM TCEP) of treatment before CTX-Clv binding became reversible. Oocytes were depolarized every 30 s and the data points were obtained at the end of a 5-s pulse. Gray and red data points correspond to the raw traces above. (b) Only one KCNE peptide reacts per CTX-Clv treatment. (Upper) CTX-sensitive current for each round. Scale bar is 0.5 μA and 0.5 s. (Lower) Chemical modification of 19% of the cell surface Q1/E1_T14C complexes with 10 μM maleimide N-ethylsulfonate, sodium salt (Mal-ES), predicts that the reversibility of CTX-Clv in the second round will be 69% if one KCNE peptide reacts per round; 88% if two KCNE are modified per round (dotted lines, which correspond to two or four KCNE subunits in the K⁺ channel complex). Washout of CTX-Clv after the second round of treatment results in 65 ± 4% (SEM) reversibility (n = 3). I/I_max is the CTX-sensitive current that was normalized before chemical treatment. Gray and red data points correspond to the CTX-sensitive currents above.