Nonspecific membrane interactions can modulate BK channel activation

Abstract

One-sentence summary: The covalent linker between BK intracellular and transmembrane domains is likely a part of the sensing apparatus that regulates the channel activation. # Equal contributions * Corresponding Authors: Jianmin Cui: Phone: (314) 935-8896; AbstractLarge-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca 2+ . The only covalent connection between the cytosolic Ca 2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue "C-linker". To determine the linker's role in BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combing atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results also suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.Widely distributed in nerve and muscle cells, large-conductance potassium (BK) channels are characterized by a large single-channel conductance (~ 100 -300 pS) (1-5) and dual activation by both intracellular Ca 2+ and membrane voltage (6-8), thus an ideal model system for understanding the gating and sensor-pore coupling in ion channels. BK channels are involved in numerous vital physiological processes including intracellular ion homeostasis and membrane excitation, and are associated with pathogenesis of many diseases such as epilepsy, stroke, autism and hypertension (9). Functional BK channels are homo-tetramers, each containing three distinct domains (Figure 1a). The voltage sensor domain (VSD) detects membrane potential, the pore-gate domain (PGD) controls the K + selectivity and permeation, and the cytosolic tail domain (CTD) senses various intracellular ligands including Ca 2+ . The VSD and the CTD also form a Mg 2+ binding site for Mg 2+ dependent activation (Yang 2008 NSMB). The tetrameric assembly of CTD domains is also referred to as the "gating ring". VSD and PGD together form the trans-membrane domain (TMD) of BK channels. Previous studies on mouse BK channels (10) and recent atomistic structures of full-length Aplysia californica BK channel (aSlo1) (11, 12) reveal that CTD of each subunit reside beneath the TMD of the neighboring subunit in a surprising domain-swapped arrangement ( Figure S1a).The only covalent connection between CTD and TMD of BK channels is a 15-residue peptide referred to as the "C-linker" (R329 to K343 in the human BK channel, hSlo1) (green in Figure 1a).This linker directly connects the pore lining S6 helices in the PGD (yellow in Figure S1b) to the Nterminus...