Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring

Abstract

Large-conductance voltage-and calcium-activated K + (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca 2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K + (RCK1 and RCK2) domains. (1)(2)(3)(4)(5)(6). This intrinsic property makes them essential players in vital physiological processes where intracellular Ca 2+ signaling is coupled to control of membrane voltage, such as neurotransmission or muscle function (7-10). Functional BK channels form tetramers of α-subunits (11, 12), each containing seven transmembrane helixes (S0-S6) and a large intracellular C-terminus region (13,14). Even though BK channels can assemble with auxiliary subunits (13,(15)(16)(17)(18), the α-subunit contains the sensors for voltage and divalent ions (4, 19). The voltage across the membrane is sensed by charged amino acids in transmembrane segments S2, S3, and S4 (20-24). Physiologically, BK channels can be modulated by intracellular Ca 2+ and Mg 2+, which are sensed by the large C-terminus end of the α-subunit. This region folds into a pseudodimer of two regulator of conductance of K + domains (RCK1 and RCK2). Crystal structures of isolated BK channel C termini revealed that the tetramers of RCK dimers form a large structure called the "gating ring" (25-27). Each RCK domain contains one high-affinity Ca 2+ binding site ( Fig. 1 A and B, red circles) (2). The first site identified, named the Ca 2+ bowl (28-30), is contained within the RCK2 domain (mainly D895 and D897 in the human BK channels), whereas another site was described in the RCK1 domain (including residues D362, D367, and E535) (2, 4, 31). Additionally, Mg 2+ ions bind specifically, but with low affinity, to a site formed by four amino acids: two from the transmembrane region (D99 and N172) and two located in the RCK1 (E374 and E399) (Fig. 1C) (2,4,32,33,42,43). This property provides a unique opportunity to assess the independent role of each high-affinity binding site in the conformational rearrangements of the gating ring associated with BK channel function. Using patch-clamp fluorometry (44) . Using FRET, we monitored the conformational changes of different regions of the gating ring upon binding of divalent ions. Our results reveal the existence of additive and independent motions by the two RCK domains that form the gating ring. These results indicate that the gating ring is a flexible structure capable of complex conformational changes that can be triggered specifically by different divalent ions.Author contributions: P.M., T.G., and M.H. designed research; P.M. performed research; P.M., T.G., and M.H. analyzed data; and P.M., T.G., and M.H. wrote the paper.The authors declare no conflict of interest.

Mechanistic insight into the heme-independent interplay between iron and carbon monoxide in CFTR and Slo1 BKCa channels

“…Because Mg 2+ is coordinated with six oxygen atoms 71 , the binding of Mg 2+ to the Ca 2+ bowl may leave N449 away from the Ca 2+ bowl 72 . In the meanwhile, the Mg 2+ -bound Ca 2+ bowl may also disrupt the interactions of R514 with E902 and Y904 possibly as a result of a cooperation between two Ca 2+ sites 52–53 .…”

“…Thus, the Ca 2+ /Mg 2+ -free Slo1 BK channel may Ca not be fully closed possibly as a result of the absence of two inhibitive Fe 2+ bridges 53 . In the presence of 10 mM Mg 2+ or 200 mM Ba 2+ , the binding of Mg 2+ or Ba 2+ to the Ca 2+ bowl, as required by six coordination oxygen atoms 71 , may expand the Ca 2+ bowl and shrink the RCK1-RCK2 linker but may not disrupt two Fe 2+ bridges 72 . In the presence of 100 μM Cd 2+ , the possible binding of Cd 2+ to H365 and D367 and E399, as Zn 2+ does 68 , may only shrink the RCK1-RCK2 linker and disrupt the first Fe 2+ bridge 71 .…”

“…However, the binding of Ca 2+ to the Ca 2+ bowl and the Ca 2+ RCK1 site may not only disrupt two Fe 2+ bridges but also enhance the swapping interactions of N449 with the Ca 2+ bowl and the non-swapping interactions of R514 with E902/Y904 so that the gating ring may be dramatically rearranged (Figure 6) 52–53,73 . In other words, the Fe 2+ bowl may contribute to the large changes in the FRET signal directly or indirectly 72–73 . In this regard, CO is expected to induce a large conformation change in the gating ring and CN − can reverse it (Figure 6).…”

“…However, at a higher Ca 2+ concentration (>10 μM), Ca 2+ bridging N449 with the Ca 2+ bowl may pull Q907 and C911 or K1030 away from the second Fe bowl so that no CO or CN − effect would be observed. On the other hand, because Mg 2+ is coordinated by 6 oxygen atoms 71 , the binding of Mg 2+ to the Ca 2+ bowl may disrupt both the swapping interaction between N449 and the Ca 2+ bowl and the non-swapping interactions of R514 with E902/Y904 as a result of the RCK1-RCK2 cooperation 52–53, 72 . In this case, neither CO nor CN − can regulate channel activity.…”

The BK channel gating ring is strongly coupled to the voltage sensor

“…Apart from Ca 2+ , it has been described that Cd 2+ selectively binds to the RCK1 site, whereas Ba 2+ and Mg 2+ show higher affinity for the RCK2 site (2,4,25,26,(28)(29)(30). Thus, intracellular concentrations of Ca 2+ , Cd 2+ , Ba 2+ or Mg 2+ can shift the voltage dependence of BK activation towards more negative potentials.…”