Bacterial Ion Channels and Their Eukaryotic Homologs

In this chapter, the author describes the Ktn domain and its role as a channel and transporter regulator, briefly considers the other bacterial systems that possess Ktn and Ktn-related domains, and, finally presents a more detailed analysis of one's understanding of the structure and operation of the KefC system. The domain can be found as an integral part of the main channel-forming protein (e.g., MthK, Kch, and KefC) as an ancillary extrinsic membrane protein (KtrA, TrkA) and as a protein attached to the membrane by a single transmembrane span (AmhM in Bacillus pseudofirmus). The protein is one of the shortest members of the family at 434 residues, truncation of the carboxy-terminal domain being responsible for the shorter length and leading to loss of the Ktn domain. Potassium uptake systems in bacteria essentially fall into three categories: K+-transporting primary ATPases, such as Kdp; secondary transporters, such as Kup; and more complex systems, e.g., TrkAEH in Escherichia coli and KtrAB in B. subtilis. The cation-proton antiports 2 (CPA2) family of proteins was originally defined around the monovalent cation-proton antiports but is now thought to contain channels as well as transporters. The analysis of the crystal structures of K+ channels has generated great insights into the mechanism of ion selectivity. It seems almost certain that the analysis of the biochemistry of channels such as MthK and KirBac will provide new insights into the gating mechanism.

Until recently, electrophysiology served as an indirect window into one's understanding of channel gating and structure. A clearer picture of protein movements involved in gating has recently emerged from the merger of crystallographic and spectroscopic studies with functional analysis. These and other emerging results are discussed from the perspective that understanding the molecular process in details of gating helps explain how a wide variety of effectors can function to open or close a target channel, allowing for the large diversity of channels. A large helix opening may not be a requirement for channel gating as a small helix bend can allow K+ ions an adequate path for flow. The cytoplasmic gating ring of the channel is formed by an octamer of RCK domains. The mechanism of channel gating lies at the core of one's understanding of how channels respond to specific stimuli. Information extracted from functional, crystallographic, and spectroscopic studies of prokaryotic channels has revealed molecular details of how the inner helices and the selectivity filter are involved in channel gating. Focusing the gating forces at a consistent position along the ion conduction pathway allows channels to exist with a large diversity of regulatory domains but maintain a conserved core architecture necessary for efficient function.

This chapter gives an overview on voltage-gated K+ channels. The introduction of molecular biology into the channel field, coupled with the ability to use patch-clamping techniques to precisely analyze properties of channels from many different types of cells, revitalized ion channel biophysics. The structure of the pore-forming domain formed by segments S5-P-S6 of the crystal structure of the full-length voltage-gated K+ channel Aeropyrum pernix (KvAP) protein was similar to that of the MthK crystal, suggesting that it was in an open conformation. The chapter discusses experiments performed on Shaker that are most informative about the structure and voltage-sensing domain. Three types of crevasse models can be envisioned. The first model implies that the transmembrane movement of S4 is relatively large and that the rest of the protein remains relatively static. The second model depicts the opposite extreme in which there is little transmembrane movement of S4, but the location of the barrier and the structures of the crevasses change. The third model is a hybrid of the other two; S4 charges move outward during activation, but this movement is accompanied by an inward movement of the transition barrier and a change in the structures of the crevasses. The first and largest category of 6-TM KK+ channel sequences has the same length of S2-S3 linkers as most eukaryotic Kv channels; this category is the most similar to eukaryotic Kv channels. According to the authors, the most closely related sequence to that of KvAP is the KvCA sequence from Clostridium acetobutylicum.

This chapter focuses on homology modeling and molecular dynamics (MD) simulations studies of ion channels for the range of single cell organisms from prokaryotes to eukaryotes. The glutamate receptor channels (GluRs) share some distant homology in their transmembrane (TM) domains with K channels but possess distinct extracellular ligand-binding domains for which several structures, of both bacterial and mammalian homologs, are known. It can be seen that molecular modeling and simulations can contribute to studies of ion channels in two respects. Modeling studies enable extrapolation from experimental structures of prokaryotic ion channels to molecular models of eukaryotic homologs, thus aiding design and interpretation of, for example, mutation experiments for dissecting structure-function relationships. Ion channel structures and ion channel models may also be used as the basis of multinanosecond MD simulations. Finally, it will become increasingly important to run multiple simulations on multiple channels to allow comparative analysis of simulation results, which in turn will enable the formulation of more general hypotheses concerning the relationship between the conformational dynamics of channel proteins and their physiological functions.

Eukaryotic voltage-sensitive sodium and calcium channels have a major structural subunit that consists of four linked, homologous domains, which contain six putative transmembrane (TM) segments for an overall total of 24. In the putative pore-lining S5-S6 linker, a glutamate residue appears in the position homologous to that acknowledged as the selectivity filter in eukaryotic sodium and calcium channels. Members of the sodium channel family typically have a selectivity ring consisting of the aspartate, glutamate, lysine, and alanine residues contributed from domains I through IV, respectively. The possibility of bacterial sodium channels being involved in rapid flagellar movement has been raised by Clapham and collaborators. Voltage-gated ion channels (VICs) are a subset of the larger P-loop ion channel family. Most voltage-activated channels exhibit two competing responses to membrane depolarization, which initiates both an activation process that results in channel opening and an inactivation process that ultimately results in channel closing. Analyses of the crystal structures of two 2-TM bacterial potassium channels led to the conclusion that the structure of KcsA represented a closed state and that of the related calcium-gated channel, MthK, crystallized in the presence of bound calcium, represented an open state. The 6-TM channels such as Shaker offer easier genetic manipulation, including the possibility of changing four residues in a functional channel for the price of a single mutation. A strong argument has been made that the bacterial sodium channels represent, or at least are closely related to, progenitors of eukaryotic channels.

Early studies of channels within Escherichia coli native membranes defined many of the fundamental properties of bacterial mechanosensitive channel (MS) channel activities. The observed activity was reported to be modulated, but not gated, by voltage, and a subsequent study demonstrated that amphipaths that intercalate into the membrane asymmetrically can modulate the sensitivity of the channel. The mscS gene family was discovered by classical genetics. The structure of E. coli MscS was solved to 3.9-Å resolution by X-ray crystallography, residues 27 to 280 were resolved. On the other hand, this study used only molecular simulation to model the permeation of the pore, an approach that inherently entertains many assumptions. As implied by some studies, the MscS extended family is not confined to E. coli. A survey of some of the MscS family members from archaea has shown researchers just how diverse the activities encoded by family members can be in conductance, sensitivity, and ionic preference; some homologs even demonstrate cationic rather than anionic preferences. The MscS-like family of channels is extremely large and diverse. In its most streamlined form, e.g., MscS in E. coli, it has many similarities with its counterpart, MscL. It appears to directly sense membrane tension, and it appears to utilize modification of transmembrane (TM) domains tilt and rotation in its gating sequence. Because the MscS and MscL families are so far removed from each other, these preserved features may reflect conserved mechanisms found in many MS channel families.

This chapter provides an overview on the role of bacterial channels in cell physiology. The proposed role of mechanosensitive (MS) channels is the rapid and nonspecific release of solutes from the cell in response to the generation of excessive turgor pressure. This model has now been tested and verified for E. coli. In most bacterial cells Cl- ions have not been predicted to have significant roles in cell physiology. In some halophilic bacteria Cl- is accumulated to provide salt balance during growth in very high salt concentrations. The aquaglyceroporins are pores that allow the permeation of water and small linear polyhydric molecules through the bacterial membrane. The cell faces two types of problems that relate to ammonium ions. First, there is the need to scavenge for ammonium when the concentration in the environment is low. Second, cells growing on broth may encounter a surfeit of cytoplasmic ammonium ions arising from the deamination of amino acids. In a wide range of bacterial genera the genes for AmtB and GlnK exist as an operon, and recent work suggests that the former is a pore that is used by cells to accelerate the passage of ammonium ions across the membrane and that GlnK is a specific component of the nitrogen-regulatory circuit that controls its activity. The increased understanding of mechanistic aspects of the channel selectivity and gating has advanced rapidly, but an appreciation of the role of channels in cell physiology lags well behind.