S. lividans pH-Gated Potassium Channel

Hardy Evans '15 and Kevin Pan '15


I. Introduction

Potassium channels allow potassium ions to diffuse rapidly across cell membranes. This diffusion plays an important role in numerous biological processes, including signal transduction, cardiac automaticity, and homeostasis. Potassium channels are tasked with combining incredibly rapid conduction rates--on the order of 10^8 ions per second--with nearly flawless selectivity. Because nearly all potassium channels face this exact challenge, their structure is highly conserved amongst different organisms and cell types. KCSA is an integral membrane protein from Streptomyces lividans with amino acid sequence similarity to all known K+ channels. Like all potassium channels, KCSA possesses highly specific architecture, particularly in its selectivity filter, the component responsible for distinguishing ligand specificity. The structural properties of KCSA are critical to its function, as they manipulate the thermodynamic and kinetics of transmembrane ion flux in a way that allows cooperation between the normally contradictory concepts of speed and accuracy.

II. General Structure

KCSA is a tetramer with four membrane-spanning . Each subunit contains two transmembrane , adjoined by the roughly 30 amino acid pore region. The pore region consists of the . The protein has four-fold symmetry about the pore. Each subunit enters the tetramer such that one transmembrane helix faces the central pore, while two outer helices face the lipid membrane. The inner helices bend slightly outwards from the pore, at an angle of 25 degrees to the membrane normal. These inner helices house the extracellular region of the pore, where the potassium selectivity filter is located. The four inner helices pack tightly against each other near the intracellular region of the protein, resembling an inverted teepee. The pore helices are inserted between the "poles" of the teepee, with the carboxy-termini pointing into the center of the channel. This particular arrangement provides multiple inter-subunit contacts, which help hold the four subunits together.

III. pH-gating

Crystallography of KCSA in the closed conformation shows a crossing of the cytosolic ends of the trans-membrane helices, blocking the pore region. During channel opening, this crossing widens in order to allow access to the pore. This widening is mediated by proton-dependent disruption of subunit interactions. At neutral pH, Glu118, Arg117 and His25 interact to create an inter- and intra-subunit network of salt bridges and hydrogen bonds . These interactions constrain the ends of the trans-membrane helices at the bundle crossing, stabilizing the closed state. At acidic pH, the glutamates are protonated and become neutral, breaking their interactions with arginine and histidine. and increasing the net positive charge on the trans-membrane helices. This destabilizes the bundle-crossing via electrostatic repulsion between the aforementioned residues. This causes the trans-membrane helices to separate, opening the channel

IV. Selectivity Filter

The selectivity filter is of a K+ channel is highly conserved amongst all species and cell types. The selectivity filter consists of the amino acid sequence TVGYG at , which is conserved in all K+ channels. The Val and Tyr side chains from the amino acid sequence point away from the pore and make specific interactions with the tilted pore helix. The four Tyr78 side chains combine with the Trp67 and 68 residues from the pore helix to form a massive sheet of that is positioned around the selectivity filter. There are four strands of sequence, contributed by each of the subunits that are arranged with their carbonyl oxygen atoms pointed inward toward the ion conduction pathway. The selectivity filter can undergo a transition involving two amide planes between and . Potassium ions diffuse through the channel at rates of approaching 10^8 ions/second under certain conditions. In order to catalyze these high diffusion rates, a K+ ion must dehydrate and cross the selectivity filter within 10 ns.

There are four binding sites in the central pore. Each binding site is a cage-structure formed by eight oxygen atoms on the vertices of a cube, each provided by the C-terminal end of a pore helix directed into the binding . These ends are associated with a significant negative end charge, due to carbonyl groups that do not form any structural interactions with the rest of the protein. These oxygen atoms "hydrate" the K+ ion, stabilizing the transition from the aqueous extracellular environment to the relatively hydrophobic intramembrane environment. Fascinatingly, the distance between the K+ ion and each oxygen is exactly equivalent to the distance between a hydrated K+ ion and a water molecule, which allows easy de-solvation of the ion. The size of each oxygen cage is such that sodium is to interact with the carbonyl oxygens, thus providing selectivity. 

In order to facilitate high conduction rates, the binding of K+ ions causes the molecule to go from a "collapsed", or "pinched", conformation to a relatively unstable conductive conformation.. The dedication of binding energy to change conformation reduces K+ affinity, increasing rate of dissociation and thus conduction. Additionally, the binding of multiple cations leads to electrostatic repulsion, which also decreases affinity and increases conduction.

V. References

1. Doyle DA, Cabral JM, Pfuetzner RA, Kuo A, Gulbis JM, et al. (1998) The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity.

2. Thompson AN, Posson DJ, Parsa PV, Nimigean CM (2008) Molecular mechanism of pH sensing in KcsA potassium channels.

3. Berneche S, Roux B (2005) A gate in the selectivity filter of potassium channels. Structure 13: 591-600.

4. Blunck R, Cordero-Morales JF, Cuello LG, Perozo E, Bezanilla F (2006) Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction. J Gen Physiol 128: 569-581.

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