C-type inactivation underlies important roles played by voltage-gated K+ (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here, we report that in voltage-gated Shaker K+ channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K+ site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K+ site, which is formed by eight carbonyl oxygen atoms, appears to be slightly too small to readily accommodate a K+ ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark characteristic of C-type inactivation.

MthK is a Ca2+-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 Ca2+-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca2+] (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 [Ca2+] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH 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 Ca2+ was not H+ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H+] raised the EC50 for Ca2+ activation by ∼4.7-fold per tenfold increase in [H+], displaying a linear relation between log(EC50) 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 Ca2+ binding and channel opening. We can account for the Ca2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca2+ allosterically activates MthK, whereas H+ opposes activation by destabilizing the binding of Ca2+.

Regulator of K þ conductance (RCK) domains control the activity of a variety of K þ transporters and channels, including the human large conductance Ca 2þ -activated K þ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca 2þ -gated K þ 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 2þ . Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca 2þ -binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca 2þ -binding sites, resulting in a stoichiometry of 24 Ca 2þ ions per channel, is consistent with the steep relation between [Ca 2þ ] and MthK channel activity. Comparison of Ca 2þ -bound and unliganded RCK domains suggests a physical mechanism for Ca 2þ -dependent conformational changes that underlie gating in this class of channels.calcium | lipid bilayer | cooperativity R egulator of K þ conductance (RCK) domains are structurally conserved ligand-binding domains that control the activity of a diverse array of K þ channels and transporters (1-3). Many prokaryotic RCK domains contain a conserved sequence motif for binding of nucleotides (NAD þ or ATP) (4, 5). In some prokaryotic and most of the known eukaryotic RCK-containing K þ channels, however, the nucleotide binding motif is absent, and these channels are modulated by cytoplasmic ions such as Na þ , H þ , or Ca 2þ (6-12).MthK is a prototypical RCK-containing K þ channel that has provided insight toward the structural basis of ion channel gating by RCK domains (2,13,14). In MthK, binding of Ca 2þ to an octameric ring of RCK domains (the gating ring), which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K þ conduction (2, 15, 16). Based on X-ray structures of the Ca 2þ -bound MthK channel and the unliganded MthK gating ring (17), it has been hypothesized that the principal Ca 2þ -dependent conformational change is initiated by the movement of a Glu side chain (E212) at a single Ca 2þ -binding site within each RCK domain ( Fig. 1A; site 1, formed by D184, E210, and E212), followed by subsequent movement of a nearby Phe side chain (F232). However, the conformational changes in the immediate vicinity of site 1 are relatively subtle compared to apparent conformational changes in other regions of the RCK domains (17); thus the mechanism by which Ca 2þ binding at site 1 modulates channel gating is unclear.To gain insight toward mechanisms underlying Ca 2þ -dependent conformational changes in the MthK RCK domain, we probed MthK structure and function using electrophysiology and crystallography. Our results demonstrate that whereas site 1 contributes energetically to Ca 2þ -dependent gating, charge-neutralization of the key Ca 2þ -coordinating residues at this site do not eliminat...

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.

DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2–/– mouse tumor xenograft model.