Counting membrane-embedded KCNE β-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 ␣-subunits with specific regulatory ␤-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of ␣-and ␤-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 ␤-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 ␤-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 ␤-subunits.membrane proteins that open and close in response to changes in membrane potential. To regulate cellular potassium flow in both excitable and nonexcitable tissues, Q1 channels coassemble with regulatory KCNE peptides, forming membraneembedded K ϩ channel complexes with various voltage-sensing and gating properties (1). The importance of forming a properly assembled Q1-KCNE complex is underscored by the mutations that give rise to long QT syndrome and congenital hearing loss (2, 3). Although the fourfold arrangement of the Q1 ␣-subunits along the ion conduction pathway is established and unquestioned, the number of KCNE ␤-subunits in the K ϩ channel complex has been a long-standing and heated debate (4-7). Goldstein and coworkers (6) have strongly argued that Q1 channels average two KCNE peptides per complex; however, they could not definitively rule out K ϩ channel complexes containing a solitary KCNE peptide. Another complicating factor is the notion that Q1 channels form complexes with KCNE ␤-subunits with multiple stoichiometries (5, 8). Thus, approaches that measure the average number of KCNE peptides in the Q1 channel complex at the cell surface are unable to readily discern between fixed and various stoichiometries.To determine the stoichiometry or stoichiometries of Q1-KCNE K ϩ channel complexes, we devised an iterative cell surface modification scheme where the labeling reagent specifically binds to the outer vestibule of the K ϩ conduction pore while covalently modifying a cysteine-bearing KCNE peptide in the channel complex (Fig. 1). Once th...

Ion channel regulation by β-secretase BACE1 – enzymatic and non-enzymatic effects beyond Alzheimer's disease

“…85 BACE1 now further complicates the assembly options and may temper with KCNQ1/KCNE1 stoichiometry, which has been vividly debated in the past years. [107][108][109][110][111] The interaction between BACE1 and KCNQ1/ KCNE1 also raises a number of clinically interesting points. For example, can the increased lethality of BACE1-deficient mice be linked to abnormal cardiac electrophysiology?…”

The genetic basis of Brugada syndrome: A mutation update

“…The KCNE3 gene consists of three exons distributed over 13 kb at chromosome 11q13-q14; only the last exon is translated [Abbott et al, 2001a]. The Kv:KCNE ion channel complexes are heterohexameric structures consisting of four a-subunits and two KCNE peptides [Morin and Kobertz, 2008]. The KCNE peptides modulate several potassium currents in the heart [Abbott and Goldstein, 2002;Abbott et al, 1999;Grunnet et al, 2003;Lewis et al, 2004;McCrossan et al, 2003;Yu et al, 2001;Zhang et al, 2001], including I Ks [Bendahhou et al, 2005], I Kr [Abbott and Goldstein, 2002], and possibly I to [Delpon et al, 2008].…”

Steric hindrance between S4 and S5 of the KCNQ1/KCNE1 channel hampers pore opening

“…On the other hand, KCNQ1 can have KCNE1 as an auxiliary subunit 10,11 . Up to four (or two) KCNE1 subunits bind to the KCNQ1 channel [12][13][14][15][16][17] and dramatically change various KCNQ1 channel properties including activation/deactivation kinetics, voltage dependence and single channel conductance 10,11,[18][19][20] . As the slowly activating KCNQ1/KCNE1 channel (also known as the I Ks channel in the heart) plays a particularly critical role in controlling cardiac excitability, how KCNE1 modulates the gating of KCNQ1 has been one of the central questions in the field.…”