KcsA potassium channel

KcsA (K channel of streptomyces A) is a prokaryotic potassium channel from the soil bacterium Streptomyces lividans that has been studied extensively in ion channel research. The pH activated protein possesses two transmembrane segments and a highly selective pore region, responsible for the gating and shuttling of K+ ions out of the cell. The amino acid sequence found in the selectivity filter of KcsA is highly conserved among both prokaryotic and eukaryotic K+ voltage channels; as a result, research on KcsA has provided important structural and mechanistic insight on the molecular basis for K+ ion selection and conduction. As one of the most studied ion channels to this day, KcsA is a template for research on K+ channel function and its elucidated structure underlies computational modeling of channel dynamics for both prokaryotic and eukaryotic species.

History KcsA was the first potassium ion channel to be characterized using X-ray crystallography by Roderick MacKinnon and his colleagues in 1998. In the years leading up to this, research on the structure of K+ channels was centered on the use of small toxin binding to reveal the location of the pore and selectivity filter among channel residues. MacKinnon's group theorized the tetrameric arrangement of the transmembrane segments, and even suggested presence of pore-forming "loops" in the filter region made of short segments of amino acids that interacted with K+ ions passing through the channel. The discovery of strong sequence homology between KcsA and other channels in the Kv family, including the Shaker protein, attracted the attention of the scientific community especially as the K+ channel signature sequence began to appear in other prokaryotic genes. The simplicity of the two transmembrane helices in KcsA, as opposed to the six in many eukaryotic ion channels, also provided a method to understand the mechanisms of K+ channels conduction at a more rudimentary level, thereby providing even great impetus for the study of KcsA.

The crystal structure of KcsA was solved by the MacKinnon group in 1998 after discovery that removal of the C-terminus cytoplasmic domain of the native protein (residues 126–158) increases the stability of crystallized samples. A model of KcsA at the 3.2 Å resolution was produced that confirmed the tetrameric arrangement of the protein around a center pore, with one helix of each subunit facing the inside axis and the other facing outwards. Three years later, a higher resolution model was produced by Morais-Cabral and Zhou after monoclonal Fab fragments were attached to KcsA crystals to further stabilize the channel. In the early 2000s, evidence for the occupation of the selectivity filter by two K+ ions during the transport process emerged, based on energy and electrostatic calculations made to model the pore region. Continued investigation of the various opened and closed, inactive and active conformations of KcsA by other imaging methods such as ssNMR and EPR have since provided even more insight into channel structure and the forces gating the switch from channel inactivation to conduction. In 2007, Riek et al. showed that the channel opening that results from titrating the ion channel from pH 7 to pH 4, corresponds to conformational changes in two regions: transition to the ion-exchanging state of the selectivity filter, and the opening of the arrangement of TM2 at the C-terminus. This model explains the ability of KcsA to simultaneously select for K+ ions while also gating electrical conductance. In 2011, the crystal structure of full length KcsA was resolved to reveal that hindrance by the previously truncated residues permits only straightforward expansion of the intercellular ion passage region of the protein. This research provides a more detailed look into the motion of separate channel regions during ion conduction. In the present day, KcsA studies are focused on using the prokaryotic channel as a model for the channel dynamics of larger eukaryotic K+ channels, including hERG.

Structure The structure of KcsA is that of an inverted cone, with a central pore running down the center made up of two transmembrane helices (the outer-helix M1 and the inner-helix M2), which span the lipid bilayer. The channel itself is a tetramer composed of four identical, single-domain subunits (each with two α-helices) arranged so that one M2 helix faces the central pore, while the other M1 helix faces the lipid membrane. The inner helices are tilted by about 25° in relation to the lipid membrane and are slightly kinked, opening up to face the outside of the cell like a flower. These two TM helices are linked by a reentrant loop, dispersed symmetrically around a common axis corresponding to the central pore. The pore region spans approximately 30 amino acid residues and can be divided into three parts: a selectivity filter near the extracellular side, a dilated water-filled cavity at the center, and a closed gate near the cytoplasmic side formed by four packed M2 helices. This architecture is found to be highly conserved in the potassium channel family in both eukaryotes and prokaryotes. The overall length of the pore is 45 Å, and its diameter varies considerably within the distinct regions of the inner tunnel. Travelling from the intracellular region outwards (bottom to top in the picture) the pore begins with a gate region formed by M2 helices at 18 Å in diameter, and then opens into a wide cavity (~10 Å across) near the middle of the membrane. In these regions, K+ ions are in contact with surrounding water molecules but when they enter the channel from the selectivity filter at the top, the cavity is so narrow that K+ ions must shed any hydrating waters in order to enter the cell. In regards to the amino acid composition of the pore-lining residues within KcsA, the side chains lining the internal pore and cavity are predominantly hydrophobic, but within the selectivity filter, polar amino acids are present that contact the dehydrated K+ ions.

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