Neuronal KCNQ2/3 channels are recruited to lipid raft microdomains by palmitoylation of BACE1

Abstract

β-Secretase 1 (β-site amyloid precursor protein [APP]-cleaving enzyme 1, BACE1) plays a crucial role in the amyloidogenesis of Alzheimer’s disease (AD). BACE1 was also discovered to act like an auxiliary subunit to modulate neuronal KCNQ2/3 channels independently of its proteolytic function. BACE1 is palmitoylated at its carboxyl-terminal region, which brings BACE1 to ordered, cholesterol-rich membrane microdomains (lipid rafts). However, the physiological consequences of this specific localization of BACE1 remain elusive. Using spectral Förster resonance energy transfer (FRET), BACE1 and KCNQ2/3 channels were confirmed to form a signaling complex, a phenomenon that was relatively independent of the palmitoylation of BACE1. Nevertheless, palmitoylation of BACE1 was required for recruitment of KCNQ2/3 channels to lipid-raft domains. Two fluorescent probes, designated L10 and S15, were used to label lipid-raft and non-raft domains of the plasma membrane, respectively. Coexpressing BACE1 substantially elevated FRET between L10 and KCNQ2/3, whereas the BACE1-4C/A quadruple mutation failed to produce this effect. In contrast, BACE1 had no significant effect on FRET between S15 probes and KCNQ2/3 channels. A reduction of BACE1-dependent FRET between raft-targeting L10 probes and KCNQ2/3 channels by applying the cholesterol-extracting reagent methyl-β-cyclodextrin (MβCD), raft-disrupting general anesthetics, or pharmacological inhibitors of palmitoylation, all supported the hypothesis of the palmitoylation-dependent and raft-specific localization of KCNQ2/3 channels. Furthermore, mutating the four carboxyl-terminal cysteines (4C/A) of BACE1 abolished the BACE1-dependent increase of FRET between KCNQ2/3 and the lipid raft–specific protein caveolin 1. Taking these data collectively, we propose that the AD-related protein BACE1 underlies the localization of a neuronal potassium channel.

Membrane Lipid Nanodomains Modulate HCN Pacemaker Channels in Nociceptor DRG Neurons

“…3). The L10 probe, validated by our earlier work, is derived from the first 10 amino acids located at the amino terminus of Lck kinase (28, 51). L10-CFP localizes to OMDs specifically, and was shown to have low FRET with human HCN1-YFP but exhibit higher FRET with the human HCN4-YFP (28).…”

“…Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) in a humidified incubator at 37°C with 5% CO 2 in tissue culture dishes (CellTreat, Pepperell, MA). Transfection was performed on 70%-90% confluent tsA-201 cells using the lipofectamine 3000 Kit (Invitrogen, Carlsbad, CA, #L30000080), following previously established protocols (51, 62).…”

“…Transfection was performed on 70%-90% confluent tsA-201 cells using the lipofectamine 3000 Kit (Invitrogen, Carlsbad, CA, #L30000080), following previously established protocols (51,62).…”

Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells

“…Using specific fluorescent markers that segregate into either raft or non-raft domains for kinetic analysis, the PtdIns(4,5)P 2 depletion and its recovery after receptor activation were faster in the lipid-raft than in the non-raft domains (Myeong et al, 2021). The PtdIns(4,5)P 2 -sensitive KCNQ2/3 channels can be recruited to lipid-raft domains by other proteins including palmitoylated β-secretase involved in the Alzheimer's disease mechanism (Dai, 2022). While we have been focused on the plasma membrane, when we speak of PtdIns(4,5)P 2 synthesis, we must consider phosphoinositide precursors and enzyme isotypes in many membranes.…”

Palmitoylation of Voltage-Gated Ion Channels

“…Kv7 channels are critical for the transduction of sympathetic stimuli, for the muffling of excessive neuronal excitability and for controlling neuronal activation by reducing excitatory postsynaptic potentials. Kv7 channels localize to membrane rafts upon interaction with palmitoylated BACE1, leading to a new gating rearrangement [ 88 , 89 ]. Finally, palmitoylation of the regulatory β4 subunit of BK channels at Cys193 promotes exit from the ER and increases surface expression, representing an additional mechanism governing BK channel functions [ 90 ].…”