Molecular Genetics and Metabolism 105 (2012) 6472

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Molecular Genetics and Metabolism

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Genetic defects in the hotspot of inwardly rectifying K + (Kir) channels and their metabolic consequences: A review
Bikash R. Pattnaik a, b, c,, Matti P. Asuma a, Ryan Spott a, De-Ann M. Pillers a, c,
a b c

Department of Pediatrics, University of Wisconsin, Madison, USA Department of Ophthalmology & Visual Sciences, University of Wisconsin, Madison, USA Eye Research Institute, University of Wisconsin, Madison, USA

a r t i c l e

i n f o

a b s t r a c t
Inwardly rectifying potassium (Kir) channels are essential for maintaining normal potassium homeostasis and the resting membrane potential. As a consequence, mutations in Kir channels cause debilitating diseases ranging from cardiac failure to renal, ocular, pancreatic, and neurological abnormalities. Structurally, Kir channels consist of two trans-membrane domains, a pore-forming loop that contains the selectivity lter and two cytoplasmic polar tails. Within the cytoplasmic structure, clusters of amino acid sequences form regulatory domains that interact with cellular metabolites to control the opening and closing of the channel. In this review, we present an overview of Kir channel function and recent progress in the characterization of selected Kir channel mutations that lie in and near a C-terminal cytoplasmic hotspot domain. The resultant molecular mechanisms by which the loss or gain of channel function leads to organ failure provide potential opportunities for targeted therapeutic interventions for this important group of channelopathies. Published by Elsevier Inc.

Article history: Received 16 August 2011 Received in revised form 11 October 2011 Accepted 12 October 2011 Available online 19 October 2011 Keywords: Kir channelopathy AndersenTawil syndrome EAST/SeSAME syndrome Phosphoinositides KCNJ Retinopathy

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Kir channel structure and the intracellular regulatory hotspot . . . Defects in Kir channel function due to mutations within the hotspot 3.1. Kir1.1 (KCNJ1) . . . . . . . . . . . . . . . . . . . . . . . 3.2. Kir2.1 (KCNJ2) . . . . . . . . . . . . . . . . . . . . . . . 3.3. Kir3.1 (KCNJ3) . . . . . . . . . . . . . . . . . . . . . . . 3.4. Kir4.1 (KCNJ10) . . . . . . . . . . . . . . . . . . . . . . 3.5. Kir5.1 (KCNJ16) . . . . . . . . . . . . . . . . . . . . . . 3.6. Kir6.2 (KCNJ11) . . . . . . . . . . . . . . . . . . . . . . 3.7. Kir 7.1 (KCNJ13) . . . . . . . . . . . . . . . . . . . . . . 4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . Authors' contributions . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 65 66 66 66 68 68 68 68 69 70 70 70 70

1. Introduction In multi-cellular organisms, cells are characterized by energetically favorable gradient moving potassium from the intracellular to the extracellular environment. Inwardly rectifying potassium-selective
Corresponding authors at: Meriter Hospital PAB2, 202 S. Park St., Madison, WI 53715, USA. Fax: + 1 608 417 6377. E-mail addresses: bikashp@pediatrics.wisc.edu (B.R. Pattnaik), pillersd@pediatrics.wisc.edu (D.-A.M. Pillers). 1096-7192/$ see front matter. Published by Elsevier Inc. doi:10.1016/j.ymgme.2011.10.004

(Kir), channels encoded by the KCNJ gene family are constitutively active and favor the inux of potassium more readily than its efux from the cells, thereby maintaining potassium homeostasis. Kir channels are also known as IRK or KCNJ channels. Fifteen mammalian KCNJ gene products have been described which result in seven distinct Kir channels [1,2]. These channels are located within the plasma membrane of most cell types, where they regulate membrane potential and potassium homeostasis (Table 1). Kir channels contribute to functions such as the repolarization of cardiac action potentials, trans-epithelial transport, and the maintenance of the voltage

B.R. Pattnaik et al. / Molecular Genetics and Metabolism 105 (2012) 6472 Table 1 Kir gene, protein and tissue distribution. Gene KCNJ1 KCNJ2 KCNJ10 KCNJ11 KCNJ13 Protein and other identiers Kir1.1, ROMK, ROMK1 Kir2.1, HHIRK1, IRK1 Kir4.1 Kir6.2, BIR Kir7.1 Chromosome location 11q24 17q23.1q24.2 1q23.2 11p15.1 2q37 Main tissue localization Kidney Heart, skeletal muscle Glia (retinal Mller cells), kidney, cochlea Beta cells, neurons, endocrine and muscle cells Retina, small intestine, stomach, kidney

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References [34] [3941] [69,70] [90] [107]

gradient across the cell membrane. These functions are achieved by regulating the opening and closing (i.e., gating) of Kir channels [2]. For this reason, genetic alterations in Kir channels underlie many of the hereditary ion channel diseases known as channelopathies, and which affect the function of multiple organ systems [3,4]. 2. Kir channel structure and the intracellular regulatory hotspot 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 poreforming, P-loop sequence that includes the selectivity lter, followed in turn by a second trans-membrane domain, and lastly, by a C-terminal cytoplasmic domain [5,6] (Fig. 1). 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 rectiers) whereas others more modestly facilitate the efux of K + (mild inward rectiers: Kir4.1 and Kir7.1) additionally (Fig. 2). The crystal structure of a eukaryotic Kir channel (Chicken Kir 2.2) showed that in the case of the strong inward rectiers, binding of polyvalent cations like Mg 2 + and polyamines to concentric rings of acidic amino acids on the inner face of the pore block K + efux out of the cell [6]. The cytoplasmic portion of the channel thus serves as a site for regulatory modications that result in the

opening or closing of the channel [6,8]. Cytoplasmic sequences of Kir channels possess multiple binding sites for intracellular regulators such as H +, Mg 2 +, ATP, phosphoinositides, membrane cholesterol, long chain acyl Coenzyme A, polyamines, and protein kinases A and C [922]. Trans-Golgi trafcking 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 currentvoltage relationship (Fig. 2) affecting cellular physiology. Phosphoinositides, e.g. PIP2, are important regulators of Kir channel function [2427]. PIP2 is found in the cytoplasmic leaet 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 [29]. 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] (Fig. 1). This cytoplasmic hotspot is dened 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 leaet at the beginning of the C-terminal cytoplasmic domain,

Fig. 1. Kir channel topology. The predicted amino acid positions of the cytoplasmic, trans-membrane, and extracellular domains of the selected Kir channel subunits. The membrane topology illustrating the localization of two cytoplasmic (N and C terminal), two transmembrane (M1 and M2), and extracellular GYG selectivity loop with reference to plasmamembrane is represented. The cytoplasmic hotspot is highlighted with sequence homology among human Kir channels compared. Highlighted amino acids within and nearby the hotspot are shown that represent disease-causing mutations.

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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 (Fig. 1), and the metabolic consequences of the resultant Kir-associated channelopathies. 3. Defects in Kir channel function due to mutations within the hotspot 3.1. Kir1.1 (KCNJ1) The KCNJ1 gene encodes Kir1.1, also known as the Renal Outer Medullary K + channel (ROMK) [1,32,33]. Kir 1.1 is localized to kidney epithelial cells [34] and plays a crucial role in reabsorbing salt via potassium recycling in the kidney at the level of the thick ascending limb (TAL) of Henle's loop [35] (Fig. 3). Kir1.1 works in conjunction with a Na +K +2Cl co-transporter (NKCC2) to ensure proper salt and water transport between cells of the TAL and the lumen of the renal tubule. Mutations in genes encoding NKCC2 and Kir1.1 result in altered function of these ion channels and are a cause of Hyperprostaglandin E syndrome, an autosomal recessive disorder also known as Bartter syndrome (Table 2). Bartter syndrome has both neonatal and classic forms, with the classic form typically presenting in school-age children. Bartter syndrome is characterized by hypokalemic alkalosis, hyperprostaglandinuria, and hypercalciuria associated with nephrocalcinosis. The neonatal form is associated with polyhydramnios which may lead to premature birth. Affected infants suffer severe postnatal salt and water losses that can lead to lifethreatening dehydration [36]. One mutation in the KCNJ1 gene that has been associated with Bartter syndrome lies within the C-terminal cytoplasmic domain of the Kir1.1 protein, slightly upstream from the hotspot, and results in a nonpolar, hydrophobic alanine at position 177 being changed to a polar threonine residue (A177T). Electrophysiological analyses show a decrease in K + conductance by the mutant channel that is either due to disrupted channel assembly or due to altered channel conformation within the pore-forming domain [37] (Fig. 3). Mutations within the hotspot of the Kir1.1 channel affect PIP2 binding but have not been associated with a disease phenotype to-date [27,31,38]. 3.2. Kir2.1 (KCNJ2) The KCNJ2 gene encodes the protein subunits of the Kir2.1 channel [1]. Kir 2.1 is highly expressed in cardiac and skeletal muscle in addition to neural tissue [39] (Fig. 3). It plays a crucial role in determining

Fig. 2. Inward rectication properties of Kir channels. Current amplitude in response to membrane voltage is shown by representative currentvoltage (IV) relationships of Kir channels with both strong inward rectiers (A. aqua trace), or mild inward rectiers illustrated (A. dark red trace). Current in the negative direction (inward current) is indicated by a downward arrow and current in the positive direction is the outward current. For strong inward rectiers, the outward current is completely blocked by intracellular factors affecting the IV relationship as compared to the persistent outward current demonstrated by mild inward rectiers. B) IV relationship model of a mildly inward rectier channel (B. dark red trace, as in A.) showing predicted changes in both inward and outward current due to either a gain-of-function (B. green trace) or loss-of-function (B. red trace) due to genetic mutation(s).

immediately following the second trans-membrane domain (Fig. 1). 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 (Table 2). 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 dened by phosphoinositide binding specicity [30]. Phosphoinositide specicity cannot be predicted by the amino acid signature of the positively charged hotspot, nor does phosphoinositide binding specicity determine the degree of inward rectication. For example, Kir2.1 is a strong inward rectier whereas Kir4.1 is a weak inward rectier, but both are highly sensitive to the regulatory effects of PIP2 [31]. The prokaryotic bacterial Kir channel KirBac 1.1 is inhibited by PIP2 and lacks the regulatory residues that are conserved in the transmembranecytoplasmic linkers of eukaryotes whose displacement upon electrostatic interaction with PIP2 gates eukaryotic Kir channels [29]. The Kir family and its modiers therefore provide a sensitive and specic partnership that contributes to the regulation of a number of metabolic pathways.

Table 2 Genetic correlation between Kir channel hotspot mutations and disease. Geneprotein KCNJ1Kir1.1 KCNJ2Kir2.1 Mutation Ala(A) 177 Thr(T) Pro(P) 186 Leu(L) Arg(R) 189 Ile(I) Thr(T) 192 Ala(A) Thr(T) 164 Ile(I) Ala(A) 167 Val(V) Arg(R) 175 Gln(Q) Cys(C) 166 Phe(F) Ile(I) 167 Leu(L) Lys(K) 170 Arg(R) Lys(K) 170 Asn(N) Arg(R) 176 Cys(C) Glu(E) 179 Ala(A) Arg(R) 162 Trp(W) Arg(R) 166 Trp(W) Disease Hyperprostaglandin E syndrome/antenatal Bartter syndrome AndersenTawil syndrome Inheritance Autosomal recessive homozygous variant Autosomal dominant References [37] [45,46] [40,45] [47,48] [72] [72] [73] [94] [96] [97] [97] [98] [102] [112] [114]

KCNJ10Kir4.1

KCNJ11Kir6.2

SeSAME syndrome (seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance) EAST syndrome (epilepsy, sensorineural deafness, tubulopathy) same as SeSAME DEND syndrome (developmental delay, epilepsy, and neonatal diabetes) gain-of-function mutation Permanent Neonatal Diabetes Mellitus (PNDM)

Autosomal recessive homozygous variant Autosomal recessive compound heterozygous Autosomal recessive Sporadic, de novo mutation heterozygous activating mutation

KCNJ13Kir7.1

Transient Neonatal Diabetes Mellitus (TNDM) Snowake vitreoretinal degeneration Leber's congenital amaurosis

Autosomal dominant Homozygous nonsense

Photoreceptors Glucose Blood

Kir6.2

Kir7.1

K+

K+

NaK2Cl

Glucose Transporter

SUR

Kir6.2

Kir7.1

RPE

No Depolarization No Depolarization

Altered K + homeostasis between RPE and photoreceptor segment

ATP/ADP

K K

K+

Choroid

Gain-of-Function Mutations Decreased ATP Sensitivity or Increased Open Probability

Insulin Pancreatic Cell

Kir2.1

Kir2.1

K+

K+

Extracellular
Altered PIP2 -binding = Abnormal Channel Function and Diminished Kir Current

Na + Exchanger

Ventricular Cardiomyocyt e

Ca 2+ Exchanger Kir4.1 Extracellular Abnormal K + homeostasis leads to cellular hyperactivity Kir4.1

Na+ K+ K+ H OH O

Ca2+

Extracellular

Kir1.1
K+ K+

Renal Tubule Lumen K+

NaK2Cl

Kir1.1 Astroglial Endfeet

Reduced NKCC2 Activity leads to Renal Dysfunction

Aquaporin 4

Disturbed Channel Assembly

OR

TAL of Henles Loop

Altered Channel Conformation

Extracellular

Fig. 3. Tissue distribution of Kir channel subunits. The tissue-specic distribution of the Kir channels suggests that they play an important role in ion homeostasis and disease. Kir channel subunits are indicated by light blue within the membrane structure. All other possible associated channels, transporters and regulatory molecules are also shown in the membrane that controls cellular physiology. Kir channel tissue distribution along with their respective physiopathology is color-coded (Kir1.1 orange; Kir2.1 blue; Kir4.1 purple; Kir6.2 green and Kir7.1 red). Abbreviations: Kir, inwardly rectifying potassium channel; SUR, regulatory suramine subunit; ATP, adenosine tri-phosphate; ADP, adenosine di-phosphate; RPE, retinal pigment epithelium; PIP2, phosphatidylinositol (4,5)-bisphosphate; TAL, thick ascending limb.

O O

K+

K+

OH OH

Interstitial/Blood Plasma

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the resting membrane potential and in controlling the duration of action potentials in excitable cells [40,41]. In cardiac myocytes, the steep inwardly rectifying K +-current via the Kir2.1 channel is responsible for the terminal, phase 3 repolarization of the action potential [42]. Loss of Kir2.1 current lengthens the ventricular action potential and prolongs the QT interval, putting affected individuals at risk of developing ventricular tachyarrhythmias [41]. Mutations within the hotspot in the KCNJ2 gene are associated with AndersenTawil syndrome (ATS) [43], a rare autosomal dominant disease [44] that is characterized by cardiac arrhythmias, periodic paralysis, short stature, and dysmorphic features that include cleft palate, low-set ears, and limb abnormalities (syndactyly, bradydactyly, clinodactyly) [41,45,46] (Fig. 3). No genotypephenotype correlations have been described between the mutations and the various clinical features of ATS. Three loss-of-function mutations have been described in the KCNJ2 gene. All three occur in and around the highly conserved PIP2-binding domain and result in an amino acid with an electron dense R-group being converted to a non-polar group. The altered electrostatic interactions between the channel and PIP2 result in abnormal channel function. Two of the mutations lie within the C-terminal cytoplasmic hotspot of the Kir2.1 protein and exhibit decreased afnity of Kir2.1 for PIP2, which may explain the loss of channel function (Table 2) [40]. KCNJ2 mutation T192A converts a polar, hydrophilic threonine to a nonpolar, hydrophobic alanine at amino acid position 192, slightly downstream from the cytoplasmic hotspot [32]. Heteromeric channels (possessing both wild-type and T192A mutant subunits) have partial levels of Kir2.1-mediated current, whereas homomeric T192A Kir2.1 channels have a complete loss of function [47]. It is likely that the clinical ndings in ATS in its most severe form are due to abolished Kir2.1PIP2 interactions and a nonfunctional channel, whereas mutations that simply result in reduced activity of Kir2.1 lead to the less severe forms of ATS [48]. When there is complete loss of Kir2.1 channel activity, an increased frequency of spontaneous action potentials is seen that is likely triggered by altered function of the Na +/Ca 2 + exchanger [48] (Fig. 3). Reduction in potassium current in ventricular myocytes leads to spontaneous ventricular activity. The cardiac arrhythmia observed in ATS results when the cardiac action potential is prolonged due to a reduction or absence of the repolarizing current normally attributed to Kir2.1 activity [49]. In skeletal muscle, reduced Kir2.1 activity depolarizes the resting membrane potential through inactivation of Na + channel function and results in the periodic paralysis experienced by some individuals with KCNJ2 mutations [49]. 3.3. Kir3.1 (KCNJ3) The KCNJ3 gene encodes the Kir3.1 channel subunit [1,50,51]. The Kir3.1 channel is a G protein-coupled inward rectier K + channel (GIRK1) [52,53] activated by serotonin, muscarinic and opioid signaling of G protein subunits [5459]. Kir3.1 channels form a heteromeric complex with other Kir3 channels [60] and are present in a variety of human tissues, including brain, heart, eye and muscle tissue [6167]. Like other Kir channels, the C-terminus of Kir3 channel containing the cytoplasmic hotspot is also proposed to interact with PIP2 to regulate channel function [68], but there have been no reports of a mutation within the hotspot that has been associated with a channelopathy. 3.4. Kir4.1 (KCNJ10) The KCNJ10 gene encodes the Kir4.1 channel subunit. Kir4.1 is expressed in glial cells of the central nervous system, Mller cells of the retina, and in cochlea [1,69,70] (Fig. 3). Kir4.1 also plays a crucial role in facilitating salt reabsorption in the distal convoluted tubule of the kidney, where it has been hypothesized to recycle potassium by

transporting salts down an electrochemical gradient from the tubular lumen into the cell, in combination with the sodiumpotassium pump (Na +/K +-ATPase) [71,72] (Fig. 3). The tissue distribution of Kir4.1 explains why loss-of-function mutations in KCNJ10 have been associated with the autosomal recessive SeSAME syndrome (Seizures, Sensorineural deafness, Ataxia, Mental retardation, and Electrolyte imbalance), also known as EAST syndrome (Epilepsy, Ataxia, Sensorineural deafness, and Tubulopathy) [7375] (Table 2). Loss of Kir4.1 channel function in the brain or spinal cord induces astrocyte depolarization, loss of K + clearance, and a reduced seizure threshold as seen in the case of reactive gliosis (Fig. 3) [76,77]. In contrast, epithelial transport abnormalities occur when Kir4.1 function is lost in the kidney or in the cochlea. Loss of cochlear function contributes to the generation of an abnormal endocochlear potential and consequent hearing loss [78]. Loss of Kir4.1 function in the kidney leads to abnormal salt reabsorption in the distal convoluted tubule which leads to serum electrolyte abnormalities [74]. Several members of the Kir channel family are expressed in the retina [79], and although there have been no clinically signicant vision abnormalities described, the mutations associated with SeSAME/EAST syndrome do result in altered retinal physiology [80]. Two SeSAME/EAST-associated mutations have been described in the KCNJ10 gene that are found immediately upstream of the C-terminal cytoplasmic hotspot (Table 2). A third mutation lies directly within the hotspot and results in a polar threonine at position 164 changing to a hydrophobic isoleucine (T164I). The isoleucine substitution prevents the formation of a hydrogen bond with the lysine residue at amino acid position 67 that likely controls both the pH and PIP2 gating [81]. The loss of the hydrogen bond alters the channel response to pH, and this results in a loss of channel function as demonstrated by patch-clamp electrophysiology [72,75,8284]. Another hotspot mutation is located within the second transmembrane domain (A167V) and is also linked with defects in channel gating [82]. Expression of this mutation leads to the autosomal recessive nding of decreased Kir channel current when compared to the wild type [72,75,8284]. Cells expressing hotspot mutation R175Q have impaired channel function as measured by reduced current, negligible inward rectication, channel-open probabilities in the 1015% range, and reduced pH and PIP2 sensitivity [73]. 3.5. Kir5.1 (KCNJ16) The KCNJ16 gene encodes the inwardly rectifying potassium channel 5.1 (Kir5.1) [1]. Kir5.1 channels inuence the function of Kir4.1 (as in kidney) [8587] or Kir4.2 [88] channels. Mutations in Kir5.1 likely regulate the overall K + conductance by affecting other Kir subunits with which they assemble [89]. No specic mutations in the hotspot region have been described that are associated with a channelopathy. 3.6. Kir6.2 (KCNJ11) The KCNJ11 gene encodes the Kir6.2 channel, also known as an ATPsensitive potassium channel (KATP) [1,2]. Kir6.2 regulates electrical signaling in a variety of cell types, including brain, heart, skeletal muscle, and the pancreas [2,90], and acts by coupling K + movement with various aspects of cellular metabolic activity [2] (Fig. 3). KATP channels are made up of four pore-forming Kir6.2 subunits and four sulfonylurea receptor (SUR) subunits which respond to the absolute concentrations of ATP and ADP in the cell [91]. Kir6.2 channels are activated by ADP and are inhibited by ATP [2]. In the brain, Kir6.2 responds to the serum glucose concentration and contributes to mechanisms that protect against seizures. In skeletal muscle, it inuences muscular tone. Kir6.2 protects against ischemic stress in the heart [92]. In pancreatic beta cells, Kir6.2 channels trigger insulin secretion when high levels of glucose in the blood

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increase the intracellular ATP concentration. The increased concentration of ATP inhibits KATP channel activity which stimulates an increase in cytosolic Ca 2 + leading to the release of insulin [93]. In contrast, when glucose levels are low, ADP concentrations increase, leading to Kir6.2 channel activation resulting in a decrease in cytosolic Ca 2 + and an inhibition of insulin release. Abnormalities in insulin secretion that are present in the newborn period are associated with defects in Kir6.2 function. The mutations fall within two functional categories: 1) loss of channel function, and 2) abnormal biosynthesis or trafcking of Kir6.2 channels resulting in absent or reduced expression of the channel at the cellular membrane [94]. In other instances, gain-of-function mutations lead to Permanent or Transient Neonatal Diabetes Mellitus (PNDM or TNDM). Developmental delay and epileptic episodes in association with Neonatal Diabetes (DEND syndrome) occur when Kir6.2 channels become less responsive to ATP inhibition, resulting in persistent hyperpolarization and decreased insulin secretion. DEND syndrome can also be the result of altered channel biosynthesis which, in the absence of ATP inhibition, increases the stability of the open state [95]. Five de novo heterozygous activating mutations have been described that occur near, and a sixth mutation is found within, the C-terminal cytoplasmic hotspot of the Kir6.2 protein (Table 2). Genotypephenotype correlations have shown that two of these mutations are linked to DEND syndrome (C166F, I167L) three of the mutations are associated with PNDM (K170R, K170N, R176C), and one mutation is associated with TNDM (E179A) (Table 2). The effect of these mutations lies in stark contrast to the effect of mutations located distal to the hotspot region (Y12X and L147P) that result in excessive insulin secretion and have been associated with Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI, nesidioblastosis) [4]. The C166F mutation in the KCNJ11 gene is associated with DEND syndrome and results in the substitution of a polar, hydrophilic cysteine residue at amino acid position 166 with a nonpolar, aromatic phenylalanine [96]. The Kir6.2 C166F mutant channel has a marked increase in the probability of being in the open state, as well as a reduced sensitivity to ATP [96]. The proband exhibited severe intrauterine growth retardation, postnatal feeding problems, and at 3 months of age was diagnosed with diabetes mellitus which was conrmed by the presence of polyuria, polydipsia, hyperglycemia and ketosis. In addition, the proband experienced seizures, hypsarrhythmia, neurologic deterioration, diffuse hypotonia and had dysmorphic features. The I167L mutation is another cause of DEND syndrome and results in the substitution of a nonpolar isoleucine with a hydrophobic leucine residue [97]. In this instance, the proband exhibited persistent hyperglycemia within hours after birth and had seizures by two weeks of age. By 3.5 years of age, the child was severely delayed, with a developmental age of 6 months. The I167L mutation increases the probability of the channel being in the open state, thereby indirectly reducing ATP inhibition which may impact normal pore gating [97]. Other mutations in or near to the hotspot region in the KCNJ11 gene also lead to Permanent Neonatal Diabetes Mellitus (PNDM). Barbetti and colleagues studied two individuals with mutations in this region, both at residue 170, that are associated with PNDM [54] (Table 2). The rst mutation, K170R, alters the basic lysine residue to a positively charged basic arginine. The second mutation, K170N, converts the basic lysine residue to a polar, hydrophilic asparagine [98]. Both probands were diagnosed with neonatal diabetes and ketoacidosis prior to 3 months of age. Although functional assays have not been performed on either the K170R or K170N mutant channels, it is known that a K170C mutation results in a nonfunctional Kir6.2 channel [98]. A third KCNJ11 mutation of interest lies directly within the hotspot region and results in the substitution of a basic arginine with a cysteine residue at amino acid position 176 (R176C) [99]. Residue 176 is involved in PIP2 binding to the KATP channel and induction of channel

opening. When PIP2 binding to the channel is decreased, it leads to channel closure and the absence of Kir6.2 activity [100102]. The proband was diagnosed with Type 1 diabetes mellitus [99]. Transient neonatal diabetes (TNDM) typically undergoes remission during infancy, with a potential for relapse in early childhood and adolescence. Most TNDM cases result from defects in imprinting at chromosome 6q24, but surprisingly mutations in the KCNJ11 gene (which is located at 11p15.1) can also lead to this transient disease [103]. TNDM mutation E179A results in a hydrophilic glutamic acid residue being changed to a hydrophobic alanine at amino acid position 179. The E179A proband exhibited reduced birth weight, diabetes diagnosed within the rst few months of life, and the remission of diabetes during infancy. Although functional assays have not been completed for this Kir6.2 mutation, the clinical manifestations suggest that mutant Kir6.2 channels in pancreatic beta-cells result in altered release of insulin [103] (Fig. 3). 3.7. Kir 7.1 (KCNJ13) The KCNJ13 gene encodes the Kir7.1 channel subunit which plays an important role in retinal physiology [1,104]. Kir7.1 channels are mildly inwardly-rectifying [105,106] and are expressed in multiple tissues, including kidney, intestine, stomach, thyroid, spinal cord, brain, and eye [107] (Fig. 3). Partnered with NKCC transporters and the Na +K +-ATPase in the retinal pigment epithelium (RPE) apical membrane [108], the Kir7.1 channels help to maintain the electrical potential necessary for driving trans-epithelial uid transport [109,110] (Fig. 3). The apical aspects of RPE cells have an abundance of Kir7.1 and interdigitate with, and help to maintain potassium homeostasis around the photoreceptor outer segments (POS). Tight regulation of these channels by membrane PIP2 [111] may contribute to the light response that is mediated by RPE cells. For example, receptor-activated (P2Y) depletion of PIP2 reduces Kir7.1 channel activity in the apical membrane. The same signaling pathway also increases intracellular Ca 2 + concentration through the activation of IP3 mediated release. A rise in intracellular Ca 2 + concentration leads to the activation of the basal membrane Cl conductance and results in a net depolarization of the RPE cell. This effect is typically recorded as a delayed light response originating within the RPE cell [110]. Thus, regulation of Kir7.1 channel function in the apical membrane is coupled to basal membrane conductance, and inuences RPE cell physiology. The R162W mutation within the bPbbb hotspot in the KCNJ13 gene converts a basic arginine residue to a bulky tryptophan at Kir7.1 amino acid position 162 and is associated with Snowake Vitreoretinal Degeneration (SVD) [112,113] (Table 2). In the rat, a similar mutation affecting Kir7.1 results in a non-selective leaky channel and might thereby lead to premature depolarization of the RPE cells [66]. The resultant lack of regulation of transport across the RPE may contribute to the deposition of cellular metabolites as debris on the retina that is visible on the clinical fundus examination of SVD patients. Our group has recently demonstrated that a human Kir7.1 mutant clone is non-functional when is ectopically expressed in a heterologous expression system. Co-expression with the wild type Kir7.1 subunit revealed that the mutant Kir7.1 subunit has a dominant-negative effect on the heteromeric Kir7.1 channel function (Pattnaik and Pillers, unpublished results). Another loss-of-function homozygous mutation in the hotspot domain, R166X, was recently identied in a patient suffering from Leber Congenital Amaurosis (LCA) [114] (Table 2). R166X results in an early stop codon and thereby produces a truncated Kir7.1 protein lacking most of the cytoplasmic C-terminal sequence. The C-terminal sequence is critical for the membrane trafcking of the translated protein [71]. The truncated protein likely does not successfully localize to the RPE membrane domain where Kir channels normally mediate potassium inux.

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4. Summary Deregulation of Kir channel function may be one of the earliest cellular events that lead to the complex multi-organ ndings that are associated with Kir channelopathies. Kir channels are typically composed of either homo- or heterotetrameric structures whose function is highly sensitive to genetic alterations in the cytoplasmic domain. Although mutations in any domain may inuence channel function, in this review we have focused solely on those mutations that lie within or near to the bPbbb cluster hotspot. The importance of the hotspot is that it plays a role as a receptor in Kir channel regulation by intracellular metabolites, such as PIP2. The C-terminal cytoplasmic hotspot domain is critical to normal Kir channel function and thereby, mutations in this region lead to altered organ function. Given the contribution of the cytoplasmic hotspot to Kir channel regulation, any interactions between the channel and its metabolic regulators could be important targets for the development of novel therapeutic interventions for Kir channelopathies. Authors' contributions BRP, MPA, and DMP wrote this manuscript. RS wrote specic sections. All authors have approved this manuscript. Acknowledgments Supported by the UW-Madison School of Medicine and Public Health, Graduate School, and the Department of Pediatrics (DMP), UW-Medical School research project (BRP), the Rebecca Meyer Brown Professorship of the UW-Eye Research Institute (Retina Research Foundation) (BRP), Meriter Hospital and the Meriter Foundation (BRP and DMP), and supported in part by grant 1UL1RR025011 to the UWICTR from NIH/NCRR and funds from the UW Stem Cell and Regenerative Medicine Center Pilot Grant Program (BRP). The authors thank Robert Gorden for graphics and Laura Hagan for editorial assistance. References
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