High-resolution structural characterization of Kir channels predicts that their protein subunits consist of an N-terminal cytoplasmic domain followed by a trans-membrane domain, then by a pore-forming, P-loop sequence that includes the selectivity filter, followed in turn by a second trans-membrane domain, and lastly, by a C-terminal cytoplasmic domain [5
] (). Four such subunits interact to form a tetramer that creates a single-pore channel. The channel may be either homo- or hetero-tetrameric [7
]. A subgroup of Kir channels conduct K+
ions into the cells most effectively (named strong inward rectifiers) whereas others more modestly facilitate the efflux of K+
(mild inward rectifiers: Kir4.1 and Kir7.1) additionally (). The crystal structure of a eukaryotic Kir channel (Chicken Kir 2.2) showed that in the case of the strong inward rectifiers, binding of polyvalent cations like Mg2+
and polyamines to concentric rings of acidic amino acids on the inner face of the pore block K+
efflux out of the cell [6
]. The cytoplasmic portion of the channel thus serves as a site for regulatory modifications that result in the opening or closing of the channel [6
]. Cytoplasmic sequences of Kir channels possess multiple binding sites for intracellular regulators such as H+
, ATP, phosphoinositides, membrane cholesterol, long chain acyl Coenzyme A, polyamines, and protein kinases A and C [9
]. Trans-Golgi trafficking and signal sequences [23
] are also found primarily in the cytoplasmic distal C-terminal sequence. Several genetic mutations have been reported to affect Kir channel conductance, either through a gain-of-function or a loss-of-function, thereby affecting potassium conductance and resulting in alterations in the current-voltage relationship () affecting cellular physiology.
Inward rectification properties of Kir channels
Phosphoinositides, e.g. PIP2
, are important regulators of Kir channel function [24
is found in the cytoplasmic leaflet of the plasma membrane. The distribution of this inositol phosphate is dynamic, and is precisely controlled by lipid kinases, phospholipases and phosphatases [28
]. D’Avanzo and colleagues have recently demonstrated that PIP2
in the eukaryotic cell membrane serves as an evolutionary adaptation for the direct activation of Kir channels by PIP2
]. A cluster of positively charged amino acid residues in the C-terminal cytoplasmic domain creates a site that supports an electrostatic interaction between the Kir channel and the PIP2
head group [24
] (). This cytoplasmic ‘hotspot’ is defined by a cluster of basic amino acids known as the bPbbb
cluster, wherein b
represents a basic amino acid residue and P
represents proline, a polar uncharged residue. This ‘hotspot’ is found near the inner plasma membrane leaflet at the beginning of the C-terminal cytoplasmic domain, immediately following the second trans-membrane domain (). Although mutations in any aspect of the protein structure may result in channel dysfunction, in this review we will focus on those reported mutations that lie in or near to the bPbbb hotspot ().
Genetic correlation between Kir channel hotspot mutations and disease.
The various members of the Kir family can be subdivided into three distinct groups based upon their sensitivity to PIP2
regulation of channel function, with low (Kir3.1 and Kir6.1), intermediate (Kir1.1 and Kir7.1) and high (Kir2.1 and 4.1) sensitivity defined by phosphoinositide binding specificity [30
]. Phosphoinositide specificity cannot be predicted by the amino acid signature of the positively charged ‘hotspot’, nor does phosphoinositide binding specificity determine the degree of inward rectification. For example, Kir2.1 is a strong inward rectifier whereas Kir4.1 is a weak inward rectifier, but both are highly sensitive to the regulatory effects of PIP2
]. The prokaryotic bacterial Kir channel KirBac 1.1 is inhibited by PIP2
and lacks the regulatory residues that are conserved in the transmembrane-cytoplasmic linkers of eukaryotes whose displacement upon electrostatic interaction with PIP2
gates eukaryotic Kir channels [29
]. The Kir family and its modifiers therefore provide a sensitive and specific partnership that contributes to the regulation of a number of metabolic pathways.
In this review, we will focus on the correlation between genetic alterations that lie within and around the cytoplasmic ‘hotspot’ cluster of positively charged residues (), and the metabolic consequences of the resultant Kir-associated channelopathies.