How do inward rectifier potassium channels work in the heart?

How do inward rectifier potassium channels work in the heart?

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Apparently in cardiomyocytes, there is an inward rectifying potassium channel that operates during phase 4 of the cardiomyocyte action potential. I have heard that despite this potassium channel being referred to as an inward rectifying channel, that in vitro the channel in fact still transports potassium from the inside to the outside of the cell. It appears that Wikipedia seems to suggest otherwise (?).

Can someone please explain what the inward rectifier potassium channel does and its role in phase 4 of the myocyte action potential?

According to Boron's medical physiology textbook:

"Although inward rectifying potassium channels pass current better in the inward than the outward direction, the membrane potential (Vm) is typically never more negative than Ek (equilibrium potential of potassium across the membrane). Thus,net inward K+ current does not occur physiologically. As a result, the activation of GIRK channels (G protein coupled inwardly rectifying potassium channel) hyperpolarizes cardiac cells by increasing K+ conductance or outward K+ current."

Gating mechanism of the cloned inward rectifier potassium channel from mouse heart

The complementary DNA encoding the inward rectifier potassium channel was cloned from the adult mouse heart by using the polymerase chain reaction. The clone had the nucleotide sequence identical to that of the IRK1 gene cloned from a mouse macrophage cell line. Northern blot analysis revealed that the transcript of this gene was mainly expressed in the ventricle, where the inward rectifier K + channel plays a predominant role in maintaining the high negative value of the resting membrane potential. The current expressed by injection of the complementary RNA of the cloned gene into Xenopus oocytes showed a marked inward rectification that depends on the driving force of K + . A region of negative slope conductance was observed in the current-voltage relationship at potentials positive to the reversal potential. When the extracellular K + concentration was raised, the increase in outward current amplitude resulted in the “crossover” of outward current voltage relations. The fast time-dependent increase in current amplitude was recorded upon membrane repolarization from a potential positive to the reversal potential. The kinetics of the time-dependent current was very similar to that of the intrinsic gating mechanism of the native cardiac inward rectifier K + channel. Our results suggest the existence of the intrinsic gating mechanism, accounting for the extent of rectification in the current-voltage relationship in the expressed channel.

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Mechanism of inward rectification

Inward rectification of Kir channels is the result of high-affinity block by endogenous polyamines, namely spermine, and magnesium ion that plug the channel pore at more positive potentials. While the principal idea of polyamine block is understood, the specific mechanisms are unknown. Thus when the membrane potential becomes less negative (depolarization), the channel is blocked and the efflux of potassium is limited. This decreased outward current (with inward current unaffected) results in more net current being passed inward than outward hence inward-rectification of the current.

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Heart rate is tightly regulated by the combined effects of the sympathetic and parasympathetic branches of the autonomic nervous system. These two branches control heart rate by stimulating different G protein-coupled receptors (GPCRs), which in turn activate ion channels that modify the electrical properties of cardiac pacemaker cells. Sympathetic stimulation accelerates heart rate through activation of beta-adrenergic receptors (βARs), which open excitatory ion channels through the stimulatory G protein (Gαs) pathway. Parasympathetic stimulation slows heart rate through activation of the muscarinic acetylcholine receptor M2 (M2Rs), which inhibits the effect of sympathetic stimulation through the inhibitory G protein (Gαi) pathway. M2Rs also release G protein beta-gamma subunits (Gβγ), which slow heart rate by activating G protein-gated inwardrectifier potassium (GIRK) channels. Interestingly, βARs also release the very same free Gβγ, but GIRK is not activated. The molecular mechanism underlying this specificity is poorly characterized. What is the molecular basis behind signaling specificity? It has been proposed that GIRK channels form a macromolecular supercomplex with Gαi-coupled receptors and G proteins, allowing released Gβγ to bind to and activate GIRK by proximity. However the evidence for the existence of the complex remains controversial. In the first part of my thesis, I challenge the supercomplex hypothesis by providing three experimental sets against the theory. First, GIRK co-localization with GPCRs shows no preference for M2Rs over β2ARs. Second, β2ARs do not activate GIRK channels even when they are co-localized. Third, neither Gαi1 nor G protein heterotrimers functionally interact with purified GIRK1/4 channels in the planar lipid bilayer system. I conclude that protein co-localization is not the underlying mechanism to explain why GIRK channels are specifically activated by Gαi-coupled receptors. I then set out to determine the molecular basis behind signaling specificity. Using electrophysiological technologies and bioluminescent resonance electron transfer (BRET) assays, I show that M2Rs catalyze release of Gβγ subunits at higher rates than β2ARs, generating higher Gβγ concentrations that activate GIRK and regulate other targets of Gβγ. The higher rate of Gβγ release is attributable to a faster GPCR-G protein association rate in M2Rs compared to β2ARs. I conclude that the activity of GIRK channels is simply determined by the efficiency of Gβγ release from GPCRs. Physiologically, only Gαi-coupled receptors can provide sufficient concentration of Gβγ to activate GIRK channels. In the second part of my thesis, I present my work on the functional characterization of Gβγ and Na+ regulation of two cardiac GIRK channels, GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. It is known that cardiac GIRK channels are composed of GIRK1/4 heterotetramers and GIRK4 homo-tetramers. However little is known about the functional difference between the two channels. Using purified proteins and the planar lipid bilayer system, I find that Na+ binding increases Gβγ affinity in GIRK4 homo-tetramers and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Molecular Structure of K Channels

Molecular biological techniques have been successful in producing detailed information about the amino acid sequences that comprise ion channel proteins. Cloning the DNA sequence that codes for a new ion channel usually proceeds either from knowledge of its protein sequence or by following its specific electrophysiologic function in a functional assay to identify the specific gene. Electrophysiologists have been instrumental in this process by first identifying a specific current in a particular cell of which a new cloned sequence may be the source.

Once a full-length clone has been identified, one of the main endeavors that follows is to understand how the discovered sequence relates to other known channels and which sequences give rise to specific functions. What has emerged from this analysis of cloned K channels is a picture of both interrelatedness and diversity.

Motifs or Structural Domains Found in Voltage-gated K Channels

Cloning of the Shaker channel provided the first opportunity to analyze the primary amino acid sequence of a K channel. At the same time, other investigators were cloning voltage-gated sodium (Na) and calcium (Ca) channels, [4,5]and it immediately became clear that the fundamental structures of all these channels were closely related. Voltage-gated Na or Ca channel proteins are comprised of a large continuous sequence that codes for four repeated elements, each of which are homologous to one Shaker protein (Figure 1A). A single one of these structural elements is believed to span the membrane six times (Figure 1B). It is now firmly established that four Shaker proteins are needed to assemble a functional voltage-gated K channel (K V ), [6]thus producing a structure similar to voltage-gated Na and Ca channels, one that has been broken up into subunits.

Figure 1. Conserved structure of K channels. The primary amino acid sequences of voltage-gated Na and Ca channels (A) display a repeated domain structure homologous to individual voltage-gated K channel subunits (B). The cylindrical segments of the amino acid chain represent alpha-helical transmembrane components of the channel protein structure. The pore or H5 domain is a highly conserved features in al K channels that occur between two conserved transmembrane segments (S5 and S6). Voltage-gated subunits also have four other membrane-spanning domains that sense changes in membrane potential (primarily the charged residues in S4). Amino (N)- and carboxy (C)-termini contribute to inactivation and modulation by small molecules (Ca ( ++ ) and ATP). Inset: view from above the plane of the membrane (extracellular side) depicting how four K channel subunits may be arranged to form a central pore and how the six transmembrane segments of each subunit are packed. Structural model as per Durell and Guy. [103]

Figure 1. Conserved structure of K channels. The primary amino acid sequences of voltage-gated Na and Ca channels (A) display a repeated domain structure homologous to individual voltage-gated K channel subunits (B). The cylindrical segments of the amino acid chain represent alpha-helical transmembrane components of the channel protein structure. The pore or H5 domain is a highly conserved features in al K channels that occur between two conserved transmembrane segments (S5 and S6). Voltage-gated subunits also have four other membrane-spanning domains that sense changes in membrane potential (primarily the charged residues in S4). Amino (N)- and carboxy (C)-termini contribute to inactivation and modulation by small molecules (Ca ( ++ ) and ATP). Inset: view from above the plane of the membrane (extracellular side) depicting how four K channel subunits may be arranged to form a central pore and how the six transmembrane segments of each subunit are packed. Structural model as per Durell and Guy. [103]

The genes coding for voltage-gated K channels are present not only in Drosophila but also in mammals, and they are recognizable as K channels by possessing a conserved signature amino acid sequence. This structure, called the “H5” or “pore” domain, is found in all K channels cloned to date and is thought to create the lining of the ion-conducting pathway. Experiments making specific mutations in this pore-forming sequence have established that it is essential for the potassium selectivity of the channel and for binding the K channel blocker TEA + . [7]Therefore, finding this signature sequence identifies a new clone as a K channel.

Another conserved feature is found in the fourth membrane spanning segment (S4). Found in all voltage-gated ion channels, including Na and Ca channels, S4 domains contain positively charged amino acid residues, allowing them to participate in “sensing” changes in membrane potential. The charged amino acids change orientation during membrane depolarization to open the ion-conducting pathway. The movement of these charged residues can even be measured as tiny gating currents.

Since the cloning of Shaker, more genes coding for a diversity of voltage-gated K channel subunits have been isolated. They have been grouped into four subfamilies based on similarity of their amino acids sequences and on whether they will coassemble with other subunits (Table 1). For example, all of the members of the shaker subfamily have more than 60% of their primary amino acid sequence identical with other shaker subunits although compared with members of the shab subfamily, shaker members are only about 40% homologous. Furthermore, shaker subunits cannot combine with shab subunits (nor with shaw or shal subfamily members) to produce functional ion channels.

Table 1. K Channel Families

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In recent years, investigators have also discovered other protein sequences without homology to voltage-gated subunits, yet can be shown to coassemble with them and modulate their function. When authentic K channels are purified from mammalian brain, large molecular complexes (molecular weight, approximately 400 kd) are isolated. These macromolecular complexes were shown to comprise eight individual proteins, representing four copies of two distinct protein types. [8]Four voltage-gated K channel subunits ([Greek small letter alpha] subunits) were found to be associated with four copies of a new protein termed [Greek small letter beta] subunits. Subsequent cloning of the genes for [Greek small letter beta] 1, [Greek small letter beta] 2, and [Greek small letter beta] 3 subunits revealed novel sequences for these hydrophilic proteins without homology to other K channels. Coexpression of [Greek small letter beta] 1 with Shaker subfamily [Greek small letter alpha] subunits appears to alter their function by accelerating the inactivation of the K + current.

Other K Channel Families

Ca ++-activated K Channels (K Ca ) Are Structurally Similar to Voltage-gated K Channels. Electrophysiologists in the 1960s identified K currents in several tissues that not only were voltage-activated but whose open probability was also governed by intracellular Ca ++ concentrations. These currents are found in neurons, muscle, and secretory cells in vertebrates and are potently inhibited by charybdotoxin, a toxin isolated from scorpion venom. The loss of this Ca ++-activated current in Drosophila muscle gives rise to the slowpoke phenotype. [9]When the gene coding for this channel was cloned, its structure was found to have many elements in common with the Shaker-type subunits, including the H5 and S4 domains but in addition, this sequence also coded for a long extension at the carboxy-terminal end of the protein that is presumed to function in sensing Ca ++ levels or for modulation by other small molecules (Figure 1B). The relationship of these channels to each other has become clearer with the new information coming from genome sequencing projects (to be discussed).

Inward Rectifying K Channels. Another K channel family, the inward rectifiers (K ir ), can be thought of as a “functional fragment” of a voltage-gated type channel (Figure 2A, left). Here, the conserved pore domain links two transmembrane sequences. [10]These membrane-spanning sequences are the only ones present in an inward rectifier sequence and are homologous to the S5 and S6 transmembrane segments of Shaker. Inward rectifiers have unique electrophysiologic properties (to be discussed) that make them essential in many tissues for stabilizing the membrane potential.

Figure 2. Transmembrane topology of inward rectifier (left) and tandem pore domain (right) K channels. At bottom is shown a cut-away section of a model of an inward rectifier K channel displaying the putative relation of the pore-lining (H5) domain with adjoining transmembrane segments. Magnesium ions and polyamines block outward K currents from the intracellular side of the ion channel.

Figure 2. Transmembrane topology of inward rectifier (left) and tandem pore domain (right) K channels. At bottom is shown a cut-away section of a model of an inward rectifier K channel displaying the putative relation of the pore-lining (H5) domain with adjoining transmembrane segments. Magnesium ions and polyamines block outward K currents from the intracellular side of the ion channel.

A model of how an inward rectifier K channel assembles within the cell membrane is shown in Figure 2B. The transmembrane segments (M1 and M2) are responsible for maintaining integration of the protein within the hydrophobic environment of the lipid membrane, whereas the H5 region is thought to form a funnel or “inverted tepee”-type structure [11]and mediates passage of K + ions selectively. Inward rectifier K channels are subject to blockade from the inside by magnesium (Mg ++ ) ions and by polyamine compounds, such as spermine and spermidine, which causes the channel to act like a one-way street, letting K + ions into the cell freely but limiting exist (see discussion of Rectification).

Tandem Pore Domain K Channels. The most recent addition to the families of K channels has been a group whose existence has only emerged in the past 3 years. The prototypic K channel of this type was found first in common baker's yeast (TOK1) and was immediately recognized as being unique because it contained two pore or H5 sequences in tandem in its primary amino acid sequence. [12]A model of how tandem pore K channels are aligned with respect to the plasma membrane is shown on the right of Figure 2A. Since TOK1 was first described, four mammalian (TWIK-1, TREK-1, TASK, and TRAAK) and one Drosophila (ORK1) tandem pore K channels have been isolated. [13-18]It appears that these channels are responsible for baseline or leak currents and that they may be the most numerous of all K channels.

How many different K channels are there likely to be expressed in the human body?Table 1lists the K channels that have been cloned to date and groups them into the structural families presented previously. An estimate of how many more channels exist is emerging from projects devoted to sequencing the entire genome of simpler organisms. Complete sequence information is now known for many viruses, for several bacteria, including Escherichia coli, Hemophilus influenzae, and Helicobacter pylori and for the single-cell eukaryote Saccharomyces cerevisiae (yeast). For “higher” organisms, more than 50% of the sequence is known for the nematode Caenorhabditis elegans and a few percent of the genome for organisms with more complex central nervous systems such as the mouse and human. C. elegans will be the first multicellular organism whose sequence is completely determined. [19]So far, more than 45 K channels, representing all four main K channel families, have been identified in the C. elegans genome. [20]This number of K channel sequences is seemingly disproportionate as the C. elegans nervous system is so simple (308 neurons total). The tandem pore domain group is the largest, with more than half of the K channels identified as being of this type. Because there are many nematode genes that have direct homologues in higher organisms, [21-23]there are expectations that the full panoply of C. elegans K channels, if not more, will also be represented in more complex nervous systems. To understand their roles in the function of excitable tissues, we next turn to consider their basic electrophysiologic behavior.


Previous studies suggested that IK1 channels in mouse hearts are formed by heteromeric assembly of Kir2.1 and Kir2.2 subunits ( Zaritsky et al. 2001 ). Since these mouse studies were complicated by potential compensatory adaptations that occur following gene ablation, we attempted to explore the molecular basis of ventricular IK1 using virus-mediated over-expression of dominant-negative Kir2 subunits. Our Western blotting results demonstrated that Kir2.1 and Kir2.2, but not Kir2.3, are expressed in rabbit ventricular myocytes, consistent with previous mRNA measurements in mouse ( Kurachi & Takahashi, 1996 ) and rat ( Nagashima et al. 2001 ). These conclusions are also similar to those based on results in guinea-pig myocytes using unitary IK1 amplitude distributions ( Liu et al. 2001 ) and in human myocardium ( Wang et al. 1998 ) showing that Kir2.1 and Kir2.2 are the primary determinants of cardiac IK1, with very little, but measurable, contributions from Kir2.3 channels.

Transfection of rabbit ventricular myocytes with Kir2.1dn, Kir2.2dn or Kir2.1dn+ Kir2.2dn reduced IK1 levels by the same amount after 72 h of culture, while Kir2.3dn over-expression had no effect. Reduction of current to the same level would not be expected if significant proportions of IK1 channels contained Kir2.1 or Kir2.2 subunits as either homotetramers or (possibly) heterotetramers with other unidentified Kirα-subunits. Heterotetrameric assembly is also consistent with our observation that blockade of IK1 by Ba 2+ was described by a single, rather than a double, binding isotherm equation since the IC50 for Ba 2+ differs by 5- to 10-fold between the Kir2.1 and Kir2.2 homomers ( Liu et al. 2001 Preisig-Muller et al. 2002 ). Similar heterotetrameric assembly of Kv4.2 and Kv4.3 occurs in the formation of mouse cardiac Ito ( Guo et al. 2002 ). In addition, Kir2.2 expression was unchanged during culture of rabbit ventricular myocytes despite large reductions in IK1 and Kir2.1 expression. Since our Western blotting results cannot distinguish between Kir2 subunits in the sarcolemma and non-sarcolemmal membranes, it appears that Kir2.1 assists in translocation of heterotetrameric IK1 channels to the surface membrane as proposed previously from transgenic mice studies ( Zaritsky et al. 2001 ). Consistent with this suggestion, Kir2.2 currents expressed in oocytes and tsA201 cells are routinely 10-fold less than Kir2.1 currents, despite equimolar levels of RNA or DNA (H. C. Cho & P. H. Backx, unpublished data) which could be related to the observation that transfer of the distal C-terminus from Kir2.2 to Kir2.1 diminished surface expression ( Ma et al. 2001 ).

It is remarkable that the biophysical properties of IK1, including blockade by Ba 2+ (i.e. the IC50 and the Hill coefficient), did not vary when IK1 was reduced following culturing or in response to transfection with Kir2.1dn and/or Kir2.2dn. This observation is similar to the absence of changes in single channel IK1 conductance or open probability reported previously during culture of rabbit myocytes ( Veldkamp et al. 1999 ). While these findings argue in favour of a fixed stoichoimetry of IK1 channels in rabbit ventricle, the relationship between channel stoichiometry and the biophysical properties of IK1 channels is likely to be complex and further studies are clearly necessary to determine the precise stoichiometry of rabbit cardiac IK1.

The degree of reduction of IK1 density in rabbit myocytes was the same after 48 or 72 h of co-transfection with AdKir2.1dn and AdKir2.2dn. Moreover, doubling the level of AdKir2.1dn and AdKir2.2dn was also unable to induce further IK1 reductions (C. Zobel, H. C. Cho & P. H. Backx, unpublished data). These observations suggest that the maximal reduction of IK1 possible in our experimental conditions is about 70 %. The molecular composition of the remaining IK1 is unclear. As a result of the finite turnover rates of endogenous IK1 channels, the remaining current might still be generated by Kir2.1 and Kir2.2 subunits, although, at first glance, this suggestion is inconsistent with the observation that the degree of current reduction was equivalent at 48 and 72 h post-transfection with AdKir2.1dn and AdKir2.2dn. It is nevertheless conceivable that a dynamic balance between new channel production and DN transgene expression is reached between 48 and 72 h, particularly if the level of DN transgene expression declines after 48 h in culture as has been shown previously ( Parks et al. 1999 Seharaseyon et al. 2000 ). Our inability to fully eliminate IK1 in myocytes using Kir2.1dn and Kir2.2dn could also arise from incomplete suppression of IK1, despite our previous studies establishing a potent dominant-negative action of Kir2.1dn in oocytes ( Cho et al. 2000 ). Consistent with this possibility, Kir2.1dn and Kir2.2dn were far less effective inhibitors of wild-type Kir2.X currents in tsA201 cells than in oocytes (authors' unpublished observations). Therefore, it is possible that other dominant-negative channel mutants of Kir2.1 and Kir2.2 (GYG to AAA) might more effectively eliminate rabbit cardiac IK1 under our experimental conditions and this deserves further investigation. Alternatively, IK1 remaining after infection with AdKir2.1dn and/or AdKir2.2dn might arise partially (or solely) from channels lacking Kir2.1 or Kir2.2 channel protein. Based on previous publications ( Liu et al. 2001 Preisig-Muller et al. 2002 ), one possible candidate for this remaining current would be Kir2.3 either alone or combined with other members of the Kir family. This possibility seems unlikely since in our studies transfection with AdKir2.3dn did not cause reductions in IK1 and Western blots did not reveal evidence of Kir2.3 expression. In addition, transfection with AdKir2.1dn and/or AdKir2.2dn did not affect the biophysical current–voltage properties, or blockade by Ba 2+ , of IK1 in the cultured rabbit myocytes, which (as mentioned already) suggests that the molecular composition of IK1 did not change in response to reductions in IK1 in these experiments.

It has previously been suggested that the decline in IK1 following culture is related to the corresponding decreases in t-tubule density induced by culture conditions ( Christe, 1999 ). This assertion is consistent with the observed localization of Kir2.1 ( Clark et al. 2001 ) and Kir2.2 ( Leonoudakis et al. 2001 ) in the t-tubules. However, this mechanism for reduced IK1 in culture would predict parallel declines in the expression levels of Kir2.1 and Kir2.2, which was not observed in our studies. Clearly, the identification of mechanisms responsible for reductions in IK1 following myocyte culturing warrants further investigation.

In conclusion, our results demonstrate that both Kir2.1 and Kir2.2 are functionally expressed in ventricular rabbit myocytes and the predominant molecular correlate of macroscopic IK1 appears to be heteromeric channels assembled from both Kir2.1 and Kir2.2 subunits.

Low Resting Membrane Potential and Low Inward Rectifier Potassium Currents Are Not Inherent Features of hiPSC-Derived Cardiomyocytes

Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (IK1). Here, IK1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. IK1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. IK1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and IK,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that IK1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs.

Keywords: I(K,ACh) I(K1) action potential duration engineered heart tissue human atrium human induced pluripotent stem cell-derived cardiomyocytes human ventricle inward rectifier K(+) current repolarization fraction resting membrane potential.

Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

This study was funded by the United States Department of Agriculture’s Agricultural Research Service (USDA-ARS) under project number: 3094-32000-042-00D.

Any mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply a recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study we have investigated the trafficking, cellular localization, and subunit interactions of a recently identified human inward rectifier potassium channel Kir2.6. This subunit was discovered as a gene that when mutated confers susceptibility to TPP, a condition characterized by muscle weakness or paralysis accompanied by hypokalemia and thyrotoxicosis (22). Here we characterize wild type Kir2.6 to understand how the typical native Kir2.6 contributes to muscle cell biology. Surprisingly, we find that despite its high sequence similarity to other Kir2.x subunits (㺘% identity with Kir2.2), Kir2.6 is very poorly trafficked to the surface membrane and instead primarily resides in the endoplasmic reticulum. Whole cell currents of Kir2.6 by itself are very small relative to other inward rectifier subunits because of its low surface expression. However, inward rectifier channels are composed of a heterotetrameric assembly of subunits, and we show that Kir2.6 readily coassembles with other Kir2.x subunits. Because of its dominant trafficking phenotype, Kir2.6 exerts an important regulatory control on the trafficking of inward rectifier channels through dominant negative retention in the ER. To our knowledge, this is the first demonstration of regulation of the trafficking of strong inward rectifier Kir2 potassium channels by dominant negative interaction with wild type channel subunits.

Localization of potassium channels in skeletal muscle is important to their function Kir2 channels play a major role in setting the cell resting potential, in controlling muscle cell excitability by determining the extent of sodium channel inactivation, and in clearing activity-dependent accumulation of K + from T-tubules. We have shown by immunocytochemistry that the major skeletal muscle Kir channels Kir2.1 and Kir2.2 are well positioned for these functions, with Kir2.1 channels located on the plasma membrane and the peripheral regions of T-tubules, and Kir2.2 located in central regions of T-tubules and neuromuscular junction ( Fig. 6 ). These results agree well with the proposed localization of inward rectifier channels based on electrophysiological studies in frog skeletal muscle (8, 12,�) and on biochemical membrane fractionation studies in rat skeletal muscle (1). Indeed a compelling case for Kir channel localization on T-tubules and plasma membrane was made by Wallinga et al. (15), who showed by modeling that maintenance of skeletal muscle excitability and excitation-contraction coupling during repetitive action potential firing requires the presence of Kir channels in these sites for clearance of K + and prevention of positive shifts in the resting membrane potential. Our results also are in agreement with studies in cardiac muscle in which T-tubule and plasma membrane localization, and their roles in K + accumulation have been demonstrated (5, 6, 10, 11).

Although Kir2.2 and Kir2.6 share more than 98% identity and are thought to have arisen from gene duplication (22) with several diversifications acquired throughout evolution, their trafficking and localization are widely different. Exogenous expression of Kir2.6 in both COS-1 cells and mouse skeletal muscle showed colocalization with the ER marker PDI, indicating that Kir2.6 is retained in the ER and trafficked poorly to the plasma membrane ( Figs. 1 , ​ ,7, 7 , and ​ and9). 9 ). In contrast Kir2.1 and Kir2.2 trafficked out of the ER through the Golgi, shown by colocalization with Golgi markers, and finally expressed on T-tubules and the plasma membrane ( Figs. 1 , ​ ,7, 7 , and ​ and10 10 and supplemental Fig. S2).

Even though Kir2.6 primarily is retained in the ER, a small proportion of the channel is trafficked to the cell surface in COS-1 cells. We speculated that this surface Kir2.6 formed functional homotetrameric channels. Indeed, electrophysiological investigation confirmed that Kir2.6 can form functional channels on the plasma membrane, in agreement with findings from Ryan et al. (22). Kir2.6 currents display characteristic inward rectification, with more pronounced inward currents at potentials negative to EK than at potentials positive to EK. However, when compared with Kir2.2, whole cell currents reveal a marked reduction in magnitude, implying that fewer channels are present on the plasma membrane.

It is possible that high expression levels and longer expression times in some cultured cells may allow Kir2.6 channels to escape cellular trafficking and quality control mechanisms and may account for larger Kir2.6 current magnitudes observed by Ryan et al. (22) in 293T cells. In mouse skeletal muscle, however, even after 7� days of expression in vivo, we found no evidence for Kir2.6 trafficking beyond the ER when Kir2.6 was expressed alone, suggesting that it is effectively retained in the ER ( Fig. 7 ). When expressed with Kir2.1 in mouse skeletal muscle, a very low level of Kir2.6 trafficked to the Golgi ( Fig. 9 ). In primate skeletal muscle, it is possible that a tissue-specific and species-specific accessory subunit or chaperone may aid the forward trafficking of Kir2.6 thus, a greater fraction of Kir2.6 might traffic to surface membranes in human skeletal muscle.

Although the Kir2.6 sequence includes all known anterograde trafficking signals that are present in other Kir2.x channels, it is still retained in the ER ( Figs. 1 , ​ ,5, 5 , ​ ,7, 7 , ​ ,9, 9 , and ​ and10). 10 ). Our studies show that a previously unidentified site, proline 156, is the most essential amino acid for surface trafficking of Kir2.2, and notably, the leucine at position 156 of Kir2.6 caused its retention in the ER ( Figs. 3 and ​ and8). 8 ). Alignment of all known human Kir family sequences to identify conserved amino acids shows that the amino acid pair cysteine 155 and proline 156 is conserved among nearly all eukaryotic Kir subunits, but a leucine is present in place of the proline in Kir2.6 ( Fig. 11 A). Prolines have an established role in α-helical turn structure and are commonly found as the first residue of an α-helix (48). Indeed, in the three-dimensional structure of Kir2.2 (Protein Data Bank file 3JYC ), proline 156 is located at a turn that forms the beginning of the M2 inner helix ( Fig. 11 B) (49). Significantly, the adjacent cysteine 155 is part of an intrasubunit disulfide with conserved cysteine 123 that is essential for channel function and that bridges the two extracellular loops in eukaryotic Kir2 channels (49,�). It has been shown that mutation of either of these conserved cysteines of Kir2.1 in one subunit of a channel tetramer is sufficient to eliminate wild type Kir2.1 currents in a dominant negative manner (50). We assume that proline 156 in Kir2.2 induces bending that initiates the M2 helix. Leucine at this position in Kir2.6 likely impacts channel conformation and may alter folding efficiency or disulfide bond formation that could result in retention of Kir2.6 in the ER.

Alignment of Kir2 family reveals a conserved proline located between the P-loop and the M2 helix. A, multisequence alignment of human Kir family members was performed with Clustal W (1.81) with Kir sequences from IUPHAR. Leucine 156 in Kir2.6 is instead a conserved proline that initiates the M2 transmembrane helix in nearly all other Kir channels (site is shown in bold text). B, two subunits of chicken Kir2.2 (Protein Data Bank code 3JYC ) are shown, with conserved proline 156 and an adjacent disulfide bond between cysteine 155 and cysteine 123 (49).

Interestingly, the absence of a conserved proline has been implicated in a loss of function of two voltage-gated potassium channel subunits. Kv6.3 and Kv8.1 are electrically silent homotetrameric channels because of retention in the ER, a phenomenon likely resulting from divergence of a proline-containing motif. Study of chimeric subunits between Kv6.3 and Kv2.1 and point mutation analysis in Kv8.1 revealed that a PXP motif, located in the S6 transmembrane domain in the gating hinge, is altered to PXT or PXA in the silent channels, respectively (53, 54) (Protein Data Bank file 2A79 ).

We have shown that Kir2.6 associates with Kir2.1 and Kir2.2 by immunoprecipitation ( Fig. 4 ), with important consequences on channel trafficking, plasma membrane abundance, and localization ( Figs. 3 , ​ ,5, 5 , ​ ,9, 9 , and ​ and10). 10 ). Kir2 channels are known to form by homotetrameric and heterotetrameric assembly of Kir2.x subunits, with the resulting channels reflecting the physiological properties of their subunits (3). However, the subunit composition, the extent of heteromultimerization, and the roles of individual subunits in different cell types are not known. Gene knock-out studies in mice showed that Kir2.1 and Kir2.2 contribute to K + currents in heart in a nonadditive manner, suggesting that these subunits can coassemble in vivo (21). In cardiac myocytes, the diversity of channel conductances, Ba 2+ block properties, and pH sensitivity also support a model in which channels are composed of homotetrameric and heterotetrameric subunits (33,�). Our data showing the overlapping distribution of endogenous Kir2.1 and Kir2.2 channels in skeletal muscle T-tubules are consistent with the idea that a fraction of these subunits coassemble in vivo in skeletal muscle ( Fig. 6 ).

Our trafficking studies, in which Kir2.6 causes ER retention of other Kir2.x subunits and colocalization of subunits, provide strong support for heteromultimeric formation of Kir2 channels in skeletal muscle, cardiac muscle cells, and COS-1 cells ( Figs. 3 , ​ ,5, 5 , ​ ,9, 9 , and ​ and10). 10 ). Kir2.6 is the dominant subunit when it forms a heteromultimer with Kir2.1 or Kir2.2 in vivo and has a stronger dominant negative trafficking effect on Kir2.2 compared with Kir2.1, suggesting that those subunits that are most similar in sequence may have a greater propensity to coassemble. A dominant negative trafficking phenomenon also is a well known explanation for disease-associated mutations in Andersen-Tawil syndrome where Kir2.1 𹒕� and Kir2.1 �� do not traffic to the plasma membrane. Instead, they are distributed inside the cell, although they retain their ability to coassemble with wild type channels (18). Bendahhou et al. (18) speculate that deletion of amino acids 95�, located in the outer M1 helix, results in protein misfolding and consequent ER retention. Coexpression of wild type Kir2.1 with Kir2.1 𹒕� or Kir2.1 �� trapped the wild type subunit at intracellular sites. Reduction in Kir2.1 expression on the plasma membrane can interfere with regulation of electrical excitability and muscle resting potential (18).

It is interesting to note that another gene encoding Kir2.5/Kir2.2v, also closely related to Kir2.2, may act as a negative regulator of Kir2.2 channel activity as well, but in that case the Kir2.5 subunit is electrically silent possibly because of alterations of the conserved GYG selectivity filter or C terminus (55).

Although our studies do not address the function of Kir2.6 mutations that are associated with TPP, we speculate that some of those mutations may alter channel trafficking, leading to changes in the magnitude of Kir2 currents on surface membrane and T-tubules. Two of the TPP-associated mutations, Kir2.6 R399X and Kir2.6 Q407X, cause truncation of the Kir2.6 C terminus and result in the absence of a putative PDZ-binding motif (22), a region known to be important for subcellular localization of Kir2 channels (7, 10, 11, 28, 30, 31).

The Kir2.6 gene contains a thyroid-responsive element that may regulate gene transcription (22) and consequently lead to changes in its protein abundance, ER retention of Kir2.x subunits, and reduction in inward rectifier currents. Thus, thyrotoxicity may alter a delicate balance of electrical activity in muscle. Modeling studies and human disease-associated mutations in the genes encoding Kir2.1 and Kir2.6 suggest that too little or too much inward rectifier current can alter the electrical excitability of muscle and lead to paralysis (15, 17, 22, 56). Because TPP patients normalize when treated with antithyroid agents (reviewed in Ref. 57), the consequences of high T3 on muscle conductance is of interest.

In summary, we suggest that Kir2.6 is a regulatory subunit that is mainly retained in the ER as a consequence of leucine 156. Association between Kir2.6 and Kir2.1 or Kir2.2 promotes ER retention of the heterotetramer. The amount of inward rectifier channels on the plasma membrane may depend on the ratio between the Kir2.1, Kir2.2, and Kir2.6 subunits. We speculate that homeostasis is maintained in healthy individuals, enabling a stable amount of cell surface channel expression. Together, our data support the concept that ER retention is an important mechanism in controlling skeletal muscle excitability.