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

Ligand binding sites within proteins can interact by allosteric mechanisms to modulate binding affinities and control protein function. Here we present crystal structures of the regulator of K+ conductance (RCK) domain from a K+ channel, MthK, which reveal the structural basis of allosteric coupling between two Ca2+ regulatory sites within the domain. Comparison of RCK domain crystal structures in a range of conformations and with different numbers of regulatory Ca2+ ions bound, combined with complementary electrophysiological analysis of channel gating, suggests chemical interactions that are important for modulation of ligand binding and subsequent channel opening.

Crystal structure of a Ba(2+)-bound gating ring reveals elementary steps in RCK domain activation.

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 Ca(2+)-gated K(+) channel, MthK, bound with Ba(2+), at 3.1 Å resolution. The Ba(2+)-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba(2+) bound at several positions. One of the Ba(2+) sites, termed C1, overlaps with a known Ca(2+)-activation site, determined by residues D184 and E210. Functionally, Ba(2+) can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg(2+) (up to 100 mM) was ineffective. Ba(2+) activation was abolished by the mutation D184N, suggesting that Ba(2+) 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.

Structure and function of multiple Ca2+-binding sites in a K+ channel regulator of K+ conductance (RCK) domain.

Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel.

MthK is a Ca(2+)-gated K(+) channel whose activity is inhibited by cytoplasmic H(+). To determine possible mechanisms underlying the channel's proton sensitivity and the relation between H(+) inhibition and Ca(2+)-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca(2+)] (ranging from nominally 0 to 30 mM) at a given [H(+)], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca(2+)] and open probability (Po), with a mean Hill coefficient (n(H)) of 9.9 +/- 0.9. Neither the maximal Po (0.93 +/- 0.005) nor n(H) changed significantly as a function of [H(+)] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca(2+) was not H(+) sensitive over pH ranging from 7.3 to 9.0. However, increasing [H(+)] raised the EC(50) for Ca(2+) activation by approximately 4.7-fold per tenfold increase in [H(+)], displaying a linear relation between log(EC(50)) and log([H(+)]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H(+) binding does not directly modulate either the channel's closed-open equilibrium or the allosteric coupling between Ca(2+) binding and channel opening. We can account for the Ca(2+) activation and proton sensitivity of MthK gating quantitatively by assuming that Ca(2+) allosterically activates MthK, whereas H(+) opposes activation by destabilizing the binding of Ca(2+).

An engineered right-handed coiled coil domain imparts extreme thermostability to the KcsA channel.

KcsA, a potassium channel from Streptomyces lividans, was the first ion channel to have its transmembrane domain structure determined by crystallography. Previously we have shown that its C-terminal cytoplasmic domain is crucial for the thermostability and the expression of the channel. Expression was almost abolished in its absence, but could be rescued by the presence of an artificial left-handed coiled coil tetramerization domain GCN4. In this study, we noticed that the handedness of GCN4 is not the same as the bundle crossing of KcsA. Therefore, a compatible right-handed coiled coil structure was identified from the Protein Data Bank and used to replace the C-terminal domain of KcsA. The hybrid channel exhibited a higher expression level than the wild-type and is extremely thermostable. Surprisingly, this stable hybrid channel is equally active as the wild-type channel in conducting potassium ions through a lipid bilayer at an acidic pH. We suggest that a similar engineering strategy could be applied to other ion channels for both functional and structural studies.

GCN4 enhances the stability of the pore domain of potassium channel KcsA.

The prokaryotic potassium channel from Streptomyces lividans, KcsA, is the first channel that has a known crystal structure of the transmembrane domain. The crystal structure of its soluble C-terminal domain, however, still remains elusive. Biophysical and electrophysiological studies have previously implicated the essential roles of the C-terminal domain in pH sensing and in vivo channel assembly. We examined this functional assignment by replacing the C-terminal domain with an artificial tetramerization domain, GCN4-LI. The expression of KcsA is completely abolished when its C-terminal domain is deleted, but it can be rescued by fusion with GCN4-LI. The secondary and quaternary structures of the hybrid channel are very similar to those of the wild-type channel according to CD and gel-filtration analyses. The thermostability of the hybrid channel at pH 8 is similar to that of the wild-type but is insensitive to pH changes. This supports the notion that the pH sensor of KcsA is located in the C-terminal domain. The result obtained in the present study is in agreement with the proposed functions of the C-terminal domain and we show that the channel assembly role of the C-terminal domain can be substituted with a non-native tetrameric motif. Because tetramerization domains are found in different families of potassium channels and their presence often enhances the expression of channels, replacement of the elusive C-terminal domains with a known tetrameric scaffold could potentially assist the expression of other potassium channels.

Characterization of the C-terminal domain of a potassium channel from Streptomyces lividans (KcsA).

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.