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In multi-cellular organisms, cells are characterized by energetically favorable gradient moving potassium from the intracellular to the extracellular environment. Inwardly rectifying potassium-selective (Kir), channels encoded by the KCNJ gene family are constitutively active and favor the influx of potassium more readily than its efflux from the cells, thus maintaining potassium homeostasis. Kir channels are also known as KCNJ or IRK channels. Fifteen mammalian KCNJ gene products have been described which result in seven distinct Kir channels. The channels are located within the plasma membrane of most cell types, where they regulate membrane potential and potassium homeostasis. Kir channels contribute to functions such as trans-epithelial transport, the repolarization of cardiac action potentials, and the maintenance of voltage gradient across the cell membrane. These functions are achieved by regulating the opening and closing of Kir channels. As a result, genetic alterations in Kir channels underlie a number of the hereditary ion channel diseases known as channelopathies, and which affect the function of multiple organ systems.
Molecular structure of Kir channels
The primary structures of Kir channel possess a common motif of two putative membrane-spanning domains (TM1 and TM2) linked by an extracellular pore-forming region (H5) and cytoplasmic amino (NH2)- and carboxy (COOH)- terminal domains (Figure 1A). The H5 region serves as the “ion-selectivity filter” that shares with other K+-selective ion channels the signature sequence T-X-G-Y(F)-G. Kir channel structures lack the S4 voltage sensor region which is conserved in voltage-gated Ca2+, Na+ and K+ channels. As a result, Kir channels are insensitive to membrane voltage and, when particular mechanisms regulating channel activity are absent, would be active at all Em. Their defining characteristic, inward rectification, turns out not to be an intrinsic function of the channel protein but a result of the block of outward K+ flux by intracellular substances such as polyamines and Mg2+. The primary structure of two transmembrane strands is insufficient to form a complete ion channel, and functional Kir channels are composed of four such subunits in a tetrameric complex (Figure 1B).
Figure 1. Molecular architecture of Kir channels
Classification of Kir channels
Up to now, 15 Kir subunit genes have been identified and classified into seven subfamilies (Kir1.x to Kir7.x). These subfamilies can be categorized into four functional groups: classical Kir channels (Kir2.x), G protein-gated Kir channels (Kir3.x), ATP-sensitive K channels (Kir6.x), and K+-transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x) (Figure 2). The simplicity and strong homology of the basic Kir channel subunit allow for both homomeric and heteromeric combinations to form functional Kir channels.
Figure 2. Kir channel phylogenetic tree
Kir channel hotspot mutations and disease
Cytoplasmic sequences of Kir channels possess multiple binding sites for intracellular regulators such as H+, Mg2+, ATP, phosphoinositides, polyamines, membrane cholesterol, long chain acyl-Coenzyme A, and protein kinases A and C. Trans-Golgi trafficking and signal sequences 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 loss-of-function or a gain-of-function or, thereby affecting potassium conductance and causing alterations in the current–voltage relationship affecting cellular physiology.
Phosphoinositides, such as PIP2, are important regulators of Kir channel function. PIP2 is found in the cytoplasmic leaflet of the plasma membrane. The distribution of this inositol phosphate is dynamic and precisely controlled by lipid kinases, phospholipases and phosphatases. D'Avanzo et al have 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. 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. Studies have reported that mutations which lie in or near to the bPbbb hotspot can result in channel dysfunction (Table 1).
Table 1. Kir channel hotspot mutations and disease.
|KCNJ1||Kir1.1||Ala(A) 177 Thr(T)||Hyperprostaglandin E syndrome/antenatal Bartter syndrome|
|KCNJ2||Kir2.1||Pro(P) 186 Leu(L)||Andersen–Tawil syndrome|
|KCNJ10||Kir4.1||Thr(T) 164 Ile(I)||SeSAME syndrome (seizures, sensorineural deafness, ataxia,mental retardation, and electrolyte imbalance)|
|Arg(R) 175 Gln(Q)||EAST syndrome (epilepsy, sensorineural deafness, tubulopathy)|
|KCNJ11||Kir6.2||Cys(C) 166 Phe(F)||DEND syndrome (developmental delay, epilepsy, and neonatal diabetes)|
|Lys(K) 170 Arg(R)||Permanent Neonatal Diabetes Mellitus (PNDM)|
|Glu(E) 179 Ala(A)||Transient Neonatal Diabetes Mellitus (TNDM)|
|KCNJ13||Kir7.1||Arg(R) 162 Trp(W)||Snowflake vitreoretinal degeneration|
|Arg(R) 166 Trp(W)||Leber's congenital amaurosis|
Kir channels have been found to be dynamically controlled by posttranslational modification including phosphorylation. The identification of the crucial involvement of PtdIns P2 in contributing to the activity of Kir channels has clarified many points underlying their functionality and physiological regulation. Gene-targeting techniques and the human genome project have greatly contributed to understanding the contributions of Kir channels to organ physiology and disease. Phenotypes of Kir knockout animals and the identification of loss-of-function and gain-of-function mutations in Kir genes not only reveal the functional significance of Kir channels in cells and tissues but also disclose their pathophysiological relevance. The evidence accumulated by these assays ensures that Kir channels are key elements that set Eres, control cell excitability, and drive epithelial transport and regulate hormone release. The last 15 years have seen enormous advances in structural biology that have clarified not only structure-based mechanisms underlying Kir channel activity but also the relationships between channel architecture, binding of diverse small substances, and regulation of the channel’s function. These experimental approaches are now being effectively combined to advance understanding of Kir channels.