Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity

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...

Section: Resultsmentioning

confidence: 99%

Structure and function of multiple Ca ²⁺ -binding sites in a K ⁺ channel regulator of K ⁺ conductance (RCK) domain

Pau

Smith

Taylor

et al. 2011

“…The antiport mechanism provides a direct mechanistic basis for the H ϩ influx that mediates electrophile resistance when GSHdependent inhibition of K ϩ efflux by KefFC is released by Based on studies of other transporters (21,(23)(24)(25), KefC is likely to be a homooligomer to which an oligomeric KefF binds on the cytoplasmic side. Antiport activity is inhibited by GSH, which probably binds to KefC.…”

Section: Discussionmentioning

confidence: 99%

“…The two Bacillus ancillary proteins, AmhM and YhaT, exhibit 28% sequence identity and 56% similarity to one another. They possess domains called either KTN (K ϩ transport nucleotide binding) or RCK (regulating the conductance of K ϩ ) (21)(22)(23)(24)(25), which are predicted to be ligandbinding. By contrast, nucleotide-binding KTN/RCK domains (26,27) are part of the membrane proteins KefC and KefB (10).…”

mentioning

confidence: 99%

Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

Fujisawa

Ito

Krulwich

2007

؉ (K ؉ ) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K ؉ efflux system from Bacillus subtilis. Here a K ؉ /H ؉ antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent K m for K ؉ was in the low mM range. The peripheral protein was required for YhaU-and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb ؉ ϷK ؉ >Li ؉ >Na ؉ . Glutathione significantly inhibited KefFC-mediated K ؉ /H ؉ antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H ؉ uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH4 ؉ , the results also imply that AmhMT catalyzes NH4 ؉ /H ؉ antiport, which would prevent net cytoplasmic H ؉ loss during NH4 ؉ efflux.

“…However, Ca 2+ can also apparently enter the pore of the channel to produce a rapid blockade of outward K + current (12,(14)(15)(16)(17)(18). To avoid potential confounds arising from blocking effects of Ca 2+ , we used Cd 2+ as an alternative agonist to activate MthK channels in our experiments (19)(20)(21). Fig.…”

Section: Rapid Blockade Of Mthk Channels By Cytoplasmic Divalent Catimentioning

confidence: 99%

Initial steps of inactivation at the K ⁺ channel selectivity filter

Thomson

Heer

Smith

et al. 2014

K + efflux through K + channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K + channel. Either Mg 2+ or Ca 2+ can reduce K + efflux through MthK channels. However, Ca 2+ , but not Mg 2+ , can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca 2+ ions can partially dehydrate, enabling selective accessibility of Ca 2+ to a site at the entry to the selectivity filter. Ca 2+ binding at the site interacts with K + ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.calcium | gating | permeation | dynamics | energetics P otassium (K + ) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K + efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K + channels are further controlled by a gating phenomenon known as C-type inactivation, in which K + conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K + channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that Ctype inactivation is associated with a conformational change near the external mouth of the K + channel pore (i.e., at the canonical K + channel selectivity filter) (3-11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K + (6). Thus, K + presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg 2+ , Ca 2+ , and Sr 2+ ) as probes of inactivation mechanisms in MthK, a model K + channel of known structure (Fig. 1) (12-14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our el...