Structural Features of Inward Rectifier K Channels

All K+ channels are tetrameric arrangements of a core pore-forming domain, consisting of two transmembrane helices, a "pore loop," and cytoplasmic N- and C-termini (20). Voltage-gated K+ (Kv) channels contain additional transmembrane helices with charged residues that sense membrane voltage, but Kir channel subunits contain only the core domain. The two transmembrane helices of the core domain (M1 and M2, corresponding to the pore-forming S5 and S6 segments of Kv channels) line the pathway of ion permeation through the membrane (2). Recent determination of atomic resolution structures for Kir3.1 (cytoplasmic domains only) and the bacterial KirBacl.1 (cytoplasmic and transmembrane domains; Fig. 1A) inward-rectifier K+ channels has illuminated many unique and interesting properties of the Kir channel pore (21,22). Kir channels exhibit the characteristic re-entrant pore loop structure that forms the selectivity filter of the channel. The selectivity filter underlies the ability of the channel to selectively permeate K+ in the presence of other physiological cations, such as Na+ (23). Atomic resolution structures of Kir3.1 and KirBacl.1 suggest that the cytoplasmic N- and C-termini of Kir channels line a wide pore that extends the ion permeation pathway well beyond the width of the plasma membrane (Fig. 1A), and also form binding sites for multiple physiological channel ligands including ATP, GPy subunits, and anionic phospholipids such as PIP2.

Fig. 1. Kir channel structure. (A) Ribbon diagram of two opposing KirBac1.1 subunits (PDB: 1P7B) (21). The channel is divided into two distinct domains, the transmembrane domain formed by the M1 and M2 helices, and the cytoplasmic domain formed by the N- and C-termini. Conserved locations of pore lining negatively charged residues (the rectification controller and cytoplasmic charged residues) are illustrated in ball-and-stick format. (B,C) Potential locations of polyamine block in the channel pore. (B) Fixed-tail model of polyamine block, in which a polyamine is stabilized by interactions with charged residues in the inner cavity and cytoplasmic domain. (C) Fixed-head model of selectivity filter binding of polyamines, in which a polyamine occupies a deep site between the selectivity filter and charged residues in the inner cavity.

Fig. 1. Kir channel structure. (A) Ribbon diagram of two opposing KirBac1.1 subunits (PDB: 1P7B) (21). The channel is divided into two distinct domains, the transmembrane domain formed by the M1 and M2 helices, and the cytoplasmic domain formed by the N- and C-termini. Conserved locations of pore lining negatively charged residues (the rectification controller and cytoplasmic charged residues) are illustrated in ball-and-stick format. (B,C) Potential locations of polyamine block in the channel pore. (B) Fixed-tail model of polyamine block, in which a polyamine is stabilized by interactions with charged residues in the inner cavity and cytoplasmic domain. (C) Fixed-head model of selectivity filter binding of polyamines, in which a polyamine occupies a deep site between the selectivity filter and charged residues in the inner cavity.

Although it is now well understood that blockade by intracellular polyamines is the underlying cause of inward rectification, the determination of Kir structures at atomic resolution has led several groups to revisit the topic of polyamine blockade, with a particular focus on the detailed structural basis for this process. For the remainder of this chapter, we discuss recent work aimed at identifying the structural determinants for high-affinity polyamine block and the detailed aspects of polyamine binding within the long Kir channel pore.

Structure-function studies have demonstrated that in some Kir channels, a negative charge present at one particular location in the pore-lining M2 helix (D172 in Kir2.1) ensures very strong rectification. This residue has been termed the rectification controller (Fig. 1A), and neutralization of this residue weakens, but may not abolish strong rectification (1). In other channels, the introduction of a negative charge at the equivalent residue or nearby residues can confer properties of strong rectification to otherwise weakly rectifying channels. Examples of this effect include the N171D mutation in Kir1.1 and the N160D or N160E mutations in Kir6.2 (24-26). Although wild-type Kir1.1 or Kir6.2 channels exhibit very low sensitivity to polyamine blockade, the introduction of negative charges in the inner cavity of these channels results in high-affinity polyamine blockade. Furthermore, Lu and MacKinnon have shown that when this residue is replaced with a positively charged lysine, channels exhibit a shallow intrinsic rectification but, more importantly, are polyamine insensitive (26). Based on sequence homology with crystallized K+ channels, such as KcsA or KirBac, it is now accepted that this residue lines the pore with its side chain directed toward the central axis (21). In the tetrameric channel assembly, these residues thus form a negatively charged ring within the inner cavity that appears to be critical for high-affinity polyamine binding.

Although the location of the rectification controller residue is generally conserved among strongly rectifying channels, there is actually not a strict structural requirement for the location of the negative charge in the inner cavity to confer strong rectification. Using Kir6.2 as a background channel, Kurata et al. demonstrated that introduction of negatively charged glutamate residues throughout the inner cavity of the channel can confer high sensitivity to polyamine blockade (27). It appears that the only requirements to confer high polyamine sensitivity are that the introduced negative charge resides within the inner cavity of the channel, and that the side chain faces toward the channel pore.

Other studies have identified negatively charged residues beyond M2, within the cytoplasmic domains of the channel (Fig. 1A), which also affect sensitivity to blockade by polyamines (28,29). These residues are E224 and E299 in Kir2.1, and neutralization mutations of either or both residues can significantly reduce Mg2+ and polyamine sensitivity. In addition, neutralization mutations of these cytoplasmic residues can alter the permeation properties of the channel, such that they exhibit weak inward rectification even in the absence of polyamines (30). Inspection of structures of the cytoplasmic domains of Kir channels shows that the tertiary protein structure brings these two residues into close proximity, and that the negatively charged side chains are likely directed toward the cytoplasmic pore of the channel (21,22). In this way, these residues form a second "ring" of charge within the Kir pore, in addition to the ring of charge at the position of the rectification controller. Importantly, most Kir channel types (whether strongly or weakly rectifying) have one or more negatively charged residues at positions equivalent to Kir2.1 residues E224 and E299. Therefore, the presence of negatively charged residues in the cytoplasmic domain is not sufficient to confer properties of strong rectification.

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