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

Abstract

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+.

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

“…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 .…”

“…The full-length MthK channel, containing a C-terminal thrombin cleavage site followed by a 6xHis-tag, was expressed in Escherichia coli XL-1 Blue cells on induction with 0.4 mM IPTG (isopropyl b-D-1-thiogalactopyranoside) 10 . Bacteria were collected and resuspended in 20 mM Tris, 100 mM KCl, pH 7.6 (Buffer A), and lysed by sonication in the presence of phenylmethylsulphonyl fluoride and a protease inhibitor cocktail (Complete EDTA-free, Roche).…”

“…Thus, RCK domains transduce ligand binding to gate transmembrane K þ flux in response to signalling events and cellular metabolism, in organisms ranging from bacteria to humans. In addition, binding of one ligand to an RCK domain can allosterically modulate binding of a second ligand, resulting in tuning of channel or transporter activity in response to cellular stimuli [9][10][11] . These allosteric interactions are key in generating a wide dynamic range for ligand activation of channels and transporters, in addition to linking multiple stimulus modalities to gate ion flux.…”

“…These allosteric interactions are key in generating a wide dynamic range for ligand activation of channels and transporters, in addition to linking multiple stimulus modalities to gate ion flux. Although allosteric interactions in the RCK domains have been described in functional terms, the structural bases for such interactions are yet unclear 10,12,13 .…”

“…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.…”