Crystal Structure of a Ba2+-Bound Gating Ring Reveals Elementary Steps in RCK Domain Activation

Abstract

SUMMARY RCK domains control activity of a variety of K+ channels and transporters through binding of cytoplasmic ligands. To gain insight toward mechanisms of RCK domain activation, we solved the structure of the RCK domain from the Ca2+-gated K+ channel, MthK, bound with Ba2+, at 3.1 Å resolution. The Ba2+-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba2+ bound at several positions. One of the Ba2+ sites, termed C1, overlaps with a known Ca2+-activation site, determined by residues D184 and E210. Functionally, Ba2+ can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg2+ (up to 100 mM) was ineffective. Ba2+ activation was abolished by the mutation D184N, suggesting that Ba2+ activates primarily through the C1 site. Our results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating.

Structural basis of allosteric interactions among Ca2+-binding sites in a K+ channel RCK domain

“…8a), the RCK domains may exist in a range of conformations, as observed in structures of the unliganded MthK gating ring 17,21 . Ca 2 þ binding at the C1 sites (in the N-lobes) would then lead to stabilization of a partially activated conformation 16,21 (Fig. 8b).…”

“…4a,b) 16,28 . Thus, in conditions where Ca 2 þ binding at C1 inhibits binding at C3, a Ca 2 þ signal may lead to Ca 2 þ binding at the C1 site and, in turn, may be sufficient for partial activation of the channel 21 . On the other hand, ionic or metabolic conditions that weaken inhibitory coupling would result in increased Ca 2 þ affinity at C3, and thus lead to greater channel activation in response to an equivalent Ca 2 þ signal.…”

“…If both E133 and E259 were negatively charged, they would electrostatically repel one another, and the inhibitory coupling would be weakened. Ca 2 þ activation of MthK is known to be greater with decreased [H þ ]; thus, this mechanism could account for a component of the H þ sensitivity of MthK gating, although other mechanisms may be involved 10,19,21 . Similar to MthK, the eukaryotic BK channel also contains RCK domains that are modulated by multiple, separate Ca 2 þ -binding sites 13,32,33 .…”

“…MthK is a prototypical K þ channel, whose crystal structure has provided insight towards mechanisms of channel gating by RCK domains 4,9,10,[14][15][16][17][18] . In MthK, binding of cytoplasmic Ca 2 þ to a tethered octameric ring of RCK domains (the 'gating ring') leads to a series of conformational changes that facilitates channel opening and K þ conduction 4,10,[19][20][21] . Here, by solving structures of mutant and wild-type (WT) RCK domains, we find that distinct Ca 2 þ activation sites near the amino and carboxy termini of the RCK domain (termed C1 and C3, respectively) are allosterically coupled to one another, to affect tuning of Ca 2 þ affinity and Ca 2 þ -dependent channel activation.…”

“…RCK domains are coloured as in a; [21][22][23] . Figure 1 illustrates that the MthK WT channel is activated by Cd 2 þ with an EC 50 of 49.1±5.0 mM (n ¼ 8).…”