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 channels are central to a wide range of biological processes, including electrical signaling, electrolyte homeostasis, and cell volume regulation (1). The activation states of several types of potassium channels and their relatives can be modulated by cytoplasmic ligands, such as G-proteins, calmodulin, or cyclic nucleotides; some of these ligands bind to modular domains that show remarkable sequence conservation across phyla. One such ligand-binding domain is the RCK 2 (regulator of conductance of potassium) or KTN (potassium transporter nucleotide-binding) domain, which is found in many prokaryotic K ϩ channels and transporters, as well as the eukaryotic BK channel (2-5). Many RCK domains contain a consensus nucleotide binding motif, such as those from the KtrA and TrkA transporter-associated proteins (6 -8). Another class of RCK domain, however, contains no apparent nucleotide binding motif but instead contains a Ca 2ϩ binding site. So far, the only two channels that have been demonstrated to contain a Ca 2ϩ -binding RCK domain are the BK channel and the prokaryotic channel MthK (Methanobacterium thermoautotrophicum potassium channel) (2, 3, 9 -11).X-ray and biochemical studies have suggested that the cytoplasmic ligand binding region of MthK is formed by an octameric ring of RCK domains, with four "tethered" RCK domains that are directly connected to the transmembrane pore and four "soluble" RCK domains (without transmembrane segments) that are docked onto the tethered RCKs (2, 12). An alternating arrangement of tethered and soluble RCK domains gives rise to an octameric ring assembly, called the "gating ring" because of its role in Ca 2ϩ -dependent gating of the MthK pore (2, 12). The soluble MthK RCK domain arises from an alternative AUG start codon within the MthK gene, just upstream from the RCK domain coding sequence. Mutation of this second start codon eliminates heterologous overexpression of the MthK RCK domain in E. coli while permitting overexpression of full-length subunits with tethered RCK domains (2). However, many RCK-containing K ϩ channels do not contain an AUG codon upstream from the RCK domain and eliminating the internal start codon does not necessarily abolish channel function in vivo (13). Thus, it has not been entirely clear whether the octameric RCK domain architecture observed with MthK is generally applicable or whether alternative mechanisms exist to generate this architecture.The presence of an apparent RCK domain within the BK channel C terminus has prompted the use of MthK as a limited structural template for BK channels (Fig. 1) (2, 3, 9 -11). This in turn has provided insight toward potential ligand activation mechanisms for BK channels. However, the correspondence between the Ca 2ϩ binding site of MthK and those of BK channels is not clear. For example, BK channels contain multiple Ca 2ϩ binding sites that operate in low (millimolar) and high (micromolar) affinity ranges (14 -16). MthK displays millimo-* This work was supported in part by National Institutes of Heal...
Potassium channels play an essential role in a wide range of biological processes, including cell volume regulation and the maintenance and control of electrical signals. With the advent of the structural era of ion channel biology, it has become critical to learn more about the functional properties of the prokaryotic channels, and this is the area in which genetic screens have become an increasingly useful approach. Here, we describe a bacteria-based complementation assay that we applied to investigate gating mutants of the prokaryotic K+ channel MthK, which was cloned from the archeon Methanobacterium thermoautotrophicum. The results demonstrated that heterologously expressed MthK is fully assembled and functional in Escherichia coli. This complementation assay should be useful in the initial identification of prokaryotic K+ channel mutants that result in altered channel function.
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