Modulation of MthK Potassium Channel Activity at the Intracellular Entrance to the Pore

Abstract

MthK 2 is a calcium-gated potassium channel for which the structure was solved to 3.3 Å resolution using x-ray methods (1). The crystal structure revealed MthK in its apparent open conformation; the pore-lining segments of the channel are splayed and receptive to the flow of permeant ions instead of forming a bundle crossing, which would sterically hinder ion conduction, as seen in the structure of the KcsA potassium channel (Fig. 1) (1-3). Each full-length MthK subunit contains two membrane-spanning segments (TM1 and TM2). The C-terminal end of TM2, in turn, is connected to a large cytoplasmic domain, called the RCK domain (1). Each MthK RCK domain contains a Ca 2ϩ -binding site. By sequence comparison and alignment, at least one apparent RCK-like domain can be found within the large cytoplasmic tail region of the mammalian maxi-K channel, and consequently MthK has served as a model to provide insight toward maxi-K channel structure and gating mechanism (1, 2, 4, 5).It was hypothesized that the force that opens the TM2 "gate" comes from a Ca 2ϩ -dependent conformational change in the RCK domains, which in turn tugs on a linker segment that is directly connected to TM2 (1). To be consistent with the principle underlying allosteric modulation of the channel, one would predict that in the Ca 2ϩ -bound conformation, the "splaying apart" of the TM2 segments (which opens the channel) is clearly energetically favored. TM2 can also presumably bend to close the channel while Ca 2ϩ is bound, just as the channel can open while the channel is not Ca 2ϩ -bound, although the closed state is favored. In principle, the intrinsic equilibrium between the two primary conformations of TM2 would not depend directly on Ca 2ϩ and could be modulated by other interactions, such as interactions among side chains near the TM2 bundle crossing, as observed with mutants of the Shaker K ϩ channel (6, 7). To look for potential interactions that may modulate the intrinsic MthK gating equilibrium, we used a bacterial complementation strategy. Using Escherichia coli strains that are deficient in K ϩ uptake, we identified a series of mutations that decreased complementation (and thus K ϩ uptake) in the strains. We then characterized the mutants using biochemical and electrophysiological assays. Our studies demonstrate that wild-type MthK is complementary in K ϩ uptake-deficient E. coli and thus supports K ϩ uptake via its open pore, whereas several mutations near the putative TM2 bundle crossing can reduce or eliminate complementation, primarily by reducing channel open probability. Because spontaneous channel opening is reduced in these mutants at very low Ca 2ϩ , as indicated by our complementation assay, and Ca 2ϩ -dependent opening is reduced at higher Ca 2ϩ , as shown in our electrophysiological recordings, our studies suggest that the negative charge at * This work was supported in part by Grant GM68523 (to B. S. R.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page...

Potassium Transport in Corynebacterium glutamicum Is Facilitated by the Putative Channel Protein CglK, Which Is Essential for pH Homeostasis and Growth at Acidic pH

“…For regulation of MthK activity, binding of Ca 2ϩ ions at Glu210, Glu212, and Asp184 of the RCK domain was described and the dynamic oligomerization of the soluble RCK proteins in dependence on Ca 2ϩ binding was proposed (19,26). In E. coli cells, however, MthK function was shown to be Ca 2ϩ independent, indicating that other parameters may be important under in vivo conditions (28). The eukaryotic SloI potassium channel contains an RCK domain as well and is activated by low internal pH values in the virtual absence of Ca 2ϩ , and histidine residues were proposed to participate in this process (3).…”

Initial steps of inactivation at the K + channel selectivity filter

“…MthK was expressed, purified, and reconstituted into proteoliposomes as described previously (15,17). Proteoliposomes were composed of Escherichia coli lipids (Avanti) that were rapidly frozen in liquid N 2 and stored at −80°C until use.…”

“…1). 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).…”

Genetic Screen for Potassium Leaky Small Mechanosensitive Channels (MscS) in Escherichia coli

“…LB2003 or TK2446). These strains cannot grow on a media with low (1-10 mM) potassium (28), but expression of potassium channels or transporters can restore their growth (29). This strategy was successful for isolation of potassium channels and transporters (30) as well as the mutations that activate potassium channels (31,32).…”