Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

Abstract

BK channels are regulated by two distinct physiological signals, transmembrane potential and intracellular Ca 2+ , each acting through independent modular sensor domains. However, despite a presumably central role in the coupling of sensor activation to channel gating, the pore-lining S6 transmembrane segment has not been systematically studied. Here, cysteine substitution and modification studies of the BK S6 point to substantial differences between BK and Kv channels in the structure and function of the S6-lined inner pore. Gating shifts caused by introduction of cysteines define a pattern and direction of free energy changes in BK S6 distinct from Shaker. Modification of BK S6 residues identifies pore-facing residues that occur at different linear positions along aligned BK and Kv S6 segments. Periodicity analysis suggests that one factor contributing to these differences may be a disruption of the BK S6 α-helix from the unique diglycine motif at the position of the Kv hinge glycine. State-dependent MTS accessibility reveals that, even in closed states, modification can occur. Furthermore, the inner pore of BK channels is much larger than that of K + channels with solved crystal structures. The results suggest caution in the use of Kv channel structures as templates for BK homology models, at least in the pore-gate domain.cysteine modification | K channels | Slo1 channels A s a unique member of the family of K + channels, large conductance, Ca 2+ -activated K + (BK or Slo1) channels are activated in a highly synergistic manner by two distinct physiological signals: depolarization and intracellular Ca 2+ . To accomplish this, each of the four α-subunits in a functional BK channel is constructed of three modular parts: a voltage-sensing domain composed of S1 to S4 transmembrane segments, a huge cytosolic domain sensing various intracellular ligands, such as Ca 2+ (1, 2), Mg 2+ (3), and heme (4, 5), and a pore-gate domain (PGD) formed by S5-pore loop-S6 to allow selective K + permeation under the regulation of transmembrane potential and [Ca 2+ ] i (6-8).The BK channel shares homology in its membrane-associated domain, including the voltage-sensing domain and PGD, with voltage-dependent K + (Kv) channels. Thus, the X-ray crystallographic structures of Kv channels (9, 10) have been widely used as an important template to guide structure-function studies of BK channels (11-13). However, the distant evolutionary relationship between BK and Kv channels (14) raises the possibility that Kv channel structure must be used with caution as a guide to BK channel structure. Sequence alignment (Fig. 1A) shows that BK channels and Kv channels differ at two critical positions in their pore-lining S6 transmembrane segments. First, there are two consecutive glycines (i.e., diglycine) at the conserved glycine hinge (15) in BK channels, but only one in the corresponding region of Kv channels. Second, whereas Kv channels share a conserved PXP motif near the cytosolic entrance to the inner pore, in BK channels there is only one prol...

Cadmium–cysteine coordination in the BK inner pore region and its structural and functional implications

“…Our previous study suggested that the narrowest part of BK inner pore is at the level of A316 (12). Therefore, the current result adds to the evidence suggesting that the selectivity filter is the most likely region to gate ion permeation in BK channels (11,12,18,25,26). Interestingly, the modest state dependence of A316C-Cd 2+ coordination is comparable to that observed at an inner pore location in the cyclic nucleotide gated channels (Fig.…”

“…Therefore, the observation that intracellular Cd 2+ accesses A316C with only a modest state dependence clearly shows that the cytosolic side of a closed BK channel does not significantly restrict access of permeant ions to the inner cavity. Our previous study suggested that the narrowest part of BK inner pore is at the level of A316 (12). Therefore, the current result adds to the evidence suggesting that the selectivity filter is the most likely region to gate ion permeation in BK channels (11,12,18,25,26).…”

“…Despite the evidence that large molecules can access the BK inner cavity in closed states, gated access of intracellularly applied Shaker ball peptide indicates that conformational changes do occur at the cytosolic end of BK channels (13). Furthermore, our previous study showed that the open-state modification of BK inner pore cysteines by MTSET is approximately two orders of magnitude faster than that in closed states (12), although the state dependence is weaker than that observed in Shaker channels (3). It is therefore important to determine the extent to which the BK cytosolic side may restrict inner pore access of K + by using a probe similar in size to K + .…”

“…For example, the large conductance, Ca 2+ -activated K + (BK or Slo1) channel shares with Kv channels a similar set of four VSDs attached to a central pore and gate domain (PGD), such that both channels are voltage dependent (9). However, unlike that of Kv channels, the inner pore of a closed BK channels is accessible to large molecules such as quaternary ammonium (QA) blockers (10,11) and methanethiosulfonate ethyltrimethylammonium (MTSET) (12), indicating that the cytosolic end of a closed BK channel cannot completely occlude K + flow; this indicates that a gate extracellular to that proposed for Kv channels is required to securely prevent K + flow in a closed BK channel. As a corollary, the underlying structural and conformational details required to couple VSD activation to channel opening may differ between BK and Kv channels.…”

“…Compared with cysteine modification by MTSET (12), Cd 2+ -Cys coordination provides two potential advantages for investigation of BK channel gating. First, Cd 2+ coordination can involve multiple residues with strict distance and geometric requirements.…”