β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel

Abstract

Large-conductance Ca 2+ -and voltage-activated K + (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α-or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.lanthanide resonance energy transfer | BK channels | β1-subunit I mportant physiological processes involve Ca 2+ entry into cells mediated by voltage-dependent Ca 2+ channels. This divalent cation influx is essential for life because it permits, for example, the adequate functioning of smooth muscle or neurosecretion to occur. Some mechanism must be put into action, however, to control Ca 2+ influx, either to dampen or to stop the physiological effects of the cytoplasmic increase in Ca 2+. In many cases, this dampening mechanism is accomplished by one of the most broadly expressed channels in mammals: the large-conductance Ca 2+ -and voltage-activated K + (BK) channel (1-3). Because there is a single gene coding for the BK channel (Slowpoke KNCMA1), channel diversity must be a consequence of alternative splicing and/or interaction with regulatory subunits. In fact, both mechanisms account for BK channel diversity, but the most dramatic changes in BK channel properties are brought about through the interaction with regulatory subunits, membrane-integral proteins, denominated BK β-subunits (β1-β4) (4-7) and the recently discovered γ-subunits (γ1-γ4) (8, 9).Structurally, the BK channel is a homotetramer of its poreforming α-subunit and is a member of the voltage-dependent potassium (Kv) channel family. Distinct from Kv channels, however, BK channel subunits are composed of seven transmembrane domains S0-S6 (10, 11). Little is known about the detailed structure of the membrane-spanning portion of the BK channel, or of the α/β1-subunit complex. Here, we used a variant of Förster resonance energy transfer (FRET), called lanthanide-based resonance energy transfer (LRET), to determine the positions of the N terminus (NT) and S0, S1, and S2 transmembrane segments of the α-subunit of the BK channel, as well as the position of the β1-subunit in the α/β1-subunit complex. LRET uses luminescent lanthanides (e.g., Tb 3+ ) as donor instead of conventional fluorophores. This technique has been successfully used to measure intramolecular distances and to...

Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET

“…Both a-LBT constructs displayed robust ionic currents and their activation by voltage was similar to that of the wild-type (wt) a, as previously reported (Fig. S1, A-C, top) (19). In addition, the g1 subunit was coexpressed with the a (wt) and each a-LBT construct to confirm that g1 was shifting the G-V curve of the a-LBT channels to the left, as the wt subunit ( Fig.…”

“…LRET/ SNPS calculates the positions of terbium ions chelated by lanthanide binding tag (LBT) motifs (18), which are strategically inserted in different positions of the protein of interest. In the case of b1, LRET/SNPS was used to determine the structural rearrangements due to the interaction between the awith b1 subunits (19). Given the tetrameric structure of BK, the LBT-labeled channel contains four LBT-Tb 3þ donors interacting with a single toxin-BODIPY acceptor.…”

“…Given the tetrameric structure of BK, the LBT-labeled channel contains four LBT-Tb 3þ donors interacting with a single toxin-BODIPY acceptor. Two alternatives strategies were used to study the (a þ b1)BK channel complex (19). In the first approach, the a-subunits were LBT-labeled to explore the Tb 3þ positions in the absence and the presence of b1 subunit.…”

The extracellular loop of the auxiliary β1-subunit is involved in the regulation of BKCa channel mechanosensitivity

“…The interpreted A, the background activity, was higher in the presence of the ␤ 1 -subunit. This is to be expected since ␤ 1 -subunits could shift the voltage sensitivity of BK Ca channels (3,4). In addition, adherence of the membrane to the glass in the gigaseal puts large resting tension on the patch dome in the absence of applied pressure (20,28) and ␤ 1 -ZERO-BK may be more sensitive to this resting tension than ZERO-BK.…”

“…The activity of the channels was higher in the presence of the ␤ 1 -subunit . This is to be expected since ␤ 1 -subunits could increase the voltage sensitivity of BK Ca channels (3,4). In addition, adherence of the membrane to the glass in the gigaseal puts large tension on the membrane in the absence of applied pressure (28), and the resting tension in the patch dome ranges from 0.5 to 4 dyn/cm (20).…”