Individual I _Ks channels at the surface of mammalian cells contain two KCNE1 accessory subunits

Abstract

KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K + channel α-subunits to form I Kslow (I Ks ) channels in the heart and ear. The number of E1 subunits in I Ks channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface I Ks channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, I Ks channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce I Ks channels with two E1 subunits and Q1 channels with no E1 subunits-channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.V oltage-gated potassium (K V ) channels include four α-subunits that form a single, central ion conduction pathway with four peripheral voltage sensors (1-3). Incorporation of accessory β-subunits modifies the function of K V channels to suit the diverse requirements of different tissues. KCNE genes encode minK-related peptides (MiRPs) (4-6), β-subunits with a single transmembrane span that assemble with a wide array of K V α-subunits (7, 8) to control surface expression, voltage dependence, and kinetics of gating transitions, unitary conductance, ion selectivity, and pharmacology of the resultant channel complexes (4, 9-15). I Kslow (I Ks ) channels in the heart and inner ear are formed by the α-subunit encoded by KCNQ1 (called Q1, K V LQT1, K V 7.1, or KCNQ1) and the β-subunit encoded by KCNE1 (called E1, mink, or KCNE1) (16,17). Inherited mutations in Q1 and E1 are associated with cardiac arrhythmia and deafness.The number of E1 subunits in I Ks channels has been a longstanding matter of disagreement. We first argued for two E1 subunits per channel based on the suppression of current by an E1 mutant (18). Subsequently, we reached the same conclusion by determining the total number of channels using radiolabeled charybdotoxin (CTX), a scorpion toxin that blocks channels when one molecule binds in the external conduction pore vestibule, and an antibody-based luminescence assay to tally E1 subunits (19). Morin and Kobertz (20) used iterative chemical linkage between CTX in the pore and E1, and they also assigned two accessory subunits to >95% of I Ks channels without gathering evidence for variation in subunit valence. Furthermore, when we formed I Ks channels from separate E1 and Q1 subunits and compared them with channels enforced via genetic encoding to contain two or four E1 subunits (19), we observed the natural I Ks channels to have the same gating attributes, small-molecule pharmacology, and CTX on and off rates (a reflection of pore vestibule structure) as channels encoded with two E1 subunits but not those with four. These findings support the conclusion that two E1 subunits are necessary, sufficient, and the normal number in I Ks channels.In contrast, others have argued that I Ks channels have va...

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

“…Supporting the conclusion that changes in O 2 lead to rapid SUMOylation of Na V 1.2, acute hypoxia recruited mCherry-SUMO1 to the cell surface only at sites where CFP-Na V 1.2 channels were already present and without altering the number of channels at the surface (Figure 8a,b and Table 2). Here, surface density and localization of CFP-Na V 1.2 and mCherry-SUMO1 subunits were assessed using TIRF and pixel-by-pixel analysis at room temperature (Manders et al, 1993), a method we have applied to quantify the surface density and co-assembly of cardiac K + channel a and b subunits (Plant et al, 2014). In ambient O 2 , just 16% of channel pixels were localized with mCherry-SUMO1 (67 ± 6/mm 2 ) and 84% were observed to be unmodified (340 ± 16/mm 2 ).…”

“…Single protein complexes at the surface of live CHO cells were identified using TIRFM as previously described (Plant et al, 2014). Cells were studied in a solution comprising (in mm): NaCl 130, KCl 4, MgCl 2 1.2, CaCl 2 2, HEPES 10, pH was adjusted to 7.4 with NaOH.…”

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?…”

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.…”