Inward rectifiers and their regulation by endogenous polyamines

Abstract

Inwardly-rectifying potassium (Kir) channels contribute to maintenance of the resting membrane potential and regulation of electrical excitation in many cell types. Strongly rectifying Kir channels exhibit a very steep voltage dependence resulting in silencing of their activity at depolarized membrane voltages. The mechanism underlying this steep voltage dependence is blockade by endogenous polyamines. These small multifunctional, polyvalent metabolites enter the long Kir channel pore from the intracellular side, displacing multiple occupant ions as they migrate to a stable binding site in the transmembrane region of the channel. Numerous structure-function studies have revealed structural elements of Kir channels that determine their susceptibility to polyamine block, and enable the steep voltage dependence of this process. In addition, various channelopathies have been described that result from alteration of the polyamine sensitivity or activity of strongly rectifying channels. The primary focus of this article is to summarize current knowledge of the molecular mechanisms of polyamine block, and provide some perspective on lingering uncertainties related to this physiologically important mechanism of ion channel blockade. We also briefly review some of the important and well understood physiological roles of polyamine sensitive, strongly rectifying Kir channels, primarily of the Kir2 family.

Keywords: channelopathy; inward rectifier; ion channel block; polyamines; potassium channels; voltage-dependent gating.

Figure 1

Diversity of natural and synthetic polyamines used to study blockade of Kir channels. Chemical structures of (A) naturally-occurring and (B–D) useful synthetic analogs of polyamine blockers of Kir channels are depicted with exemplar accompanying references.

Figure 2

Modular architecture of Kir channels, and the location of residues essential for polyamine block. (A) Overall structural arrangement of the transmembrane (TMD) and cytoplasmic (CTD) domains of Kir channels. All structural models are constructed based on the Kir2.2 channel co-crystallized with di-C8-PIP2 (Hansen et al., 2011). Highlighted residues are D172, E224, F254, D255, D259, and E299 [labeled in more detail in (C,D)]. (B) Expanded view of the selectivity filter region, highlighting the disulfide bond (green) and salt bridge (cyan) that are conserved among Kir channels. (C) Expanded view of the transmembrane domain (TMD), with the “rectification controller” (Kir2.1 D172) residue, and bundle crossing constriction (M183) highlighted. (D) Expanded view of the cytoplasmic domain (CTD), where two rings of charge have been identified (lower ring: F254, D255, D259; upper ring: E224, E299, Kir2.1 numbering). Residue M301 is highlighted in cyan as a recently identified position that is mutated in a familial form of SQT3.

Figure 3

Schematic diagram of polyamine migration and coupled ion movement. Spermine and other polyamines migrate through the channel pore toward a binding site in the TMD, and displace occupant ions ahead of them in the pore. The first binding step involves a low affinity interaction between spermine and various residues in the CTD. The second “deep” binding step involves polyamine migration from the cytoplasmic pore into the channel inner cavity, with a steeper voltage dependence (likely because of displacement of a greater number of ions—note that the specific arrangement of ions relative to the blocker is unknown).

Figure 4

Contrasting models of the deep spermine binding site in Kir2.1. (left panel) The “shalllow model” proposes a binding region “below” the rectification controller position, highlighted in yellow, along with residues in this region that have been demonstrated to reduce polyamine blocker affinity. (right panel) The “deep” model proposes a binding site highlighted in cyan, supported by experiments testing the interactions between polyamine block and modification of substituted pore-lining cysteines.

Figure 5

Blocker trapping approaches highlight a deep polyamine binding site. This image summarizes the effects of MTS modification of pore-lining cysteines on kinetics of polyamine binding and unbinding, highlighted by two recent studies (Kurata et al., 2010, 2013). At positions deep in the pore (red band), the potency of all tested blockers is significantly reduced after modification with positively charged MTS reagents (“clash”). At intermediate positions (blue and yellow bands), short polyamines could be “trapped” in the inner cavity by modification just below the rectification controller position (inner cavity modifier), while blockade by long polyamine analogs is dramatically disrupted because the charged modifying reagent clashes with longer blockers. At the most superficial modification position tested (green band), both the long and short polyamines can be trapped. “RC” indicates the pore depth of the “rectification controller” position.

Figure 6

Does charybdotoxin binding reveal details of amine interactions with the selectivity filter? (A) Depiction of a previously published molecular model of Kir2.1 with a polyamine bound in a deep site in the Kir inner cavity (Kurata et al., 2008). The trailing end of the polyamine is anchored around the “rectification controller” position, while the leading end engages with the most superficial intracellular aspect of the selectivity filter. Docking simulations were carried out using Autodock, and multiple molecular models of Kir2.1 (depicted) or Kir6.2[N160D], based on the KirBac1.1 crystal structure. (B) Recent crystal structure of charybdotoxin in complex with Kv1.2, illustrating an essential lysine interacting with the most superficial extracellular aspect of the selectivity filter. Both spermine and the occluding lysine side chain have similar functional groups (protonatable amines) at their termini, and both blocker types are particularly sensitive to ion concentrations on the “trans” side.