Regulation of ion channel/neurotransmitter receptor function by RNA editing

RNA editing by select adenosine deamination (A-to-I editing) alters functional determinants in certain ion channels and neurotransmitter receptors in vertebrates and invertebrates. In most cases, edited and unedited versions of a given receptor/channel co-exist to expand the functional space of the receptor population. Recent studies have characterized K(+) channels in squid that are edited at multiple positions, revealed a role for Q/R site editing in AMPA receptor assembly, and demonstrated a link between serotonin levels and the extent of editing of a mammalian serotonin receptor.     

Characterization of Five RNA Editing Sites in Shab Potassium Channels

RNA editing revises the genetic code at precise locations, creating single base changes in mRNA. These changes can result in altered coding potential and modifications to protein function. Sequence analysis of the Shab potassium channel of Drosophila melanogaster revealed five such RNA editing sites. Four are constitutively edited (I583V, T643A, Y660C and I681V) and one undergoes developmentally regulated editing (T671A). These sites are located in the S4, S5-S6 loop and the S6 segments of the channel. We examined the biophysical consequences of editing at these sites by creating point mutations, each containing the genomic (unedited) base at one of the five sites in the background of a channel in which all other sites are edited. We also created a completely unedited construct. The function of these constructs was characterized using two-microelectrode voltage clamp in Xenopus oocytes. Each individual 'unediting' mutation slowed the time course of deactivation and the rise time during channel activation. Two of the mutants exhibited significant hyperpolarized shifts in their midpoints of activation. Constructs that deactivated slowly also inactivated slowly, supporting a mechanism of closed-state inactivation. One of the editing sites, position 660, aligns with the Shaker 449 residue, which is known to be important in tetraethylammonium (TEA) block. The aromatic, genomically-encoded residue tyrosine at this position in Shab enhances TEA block 14 fold compared to the edited residue, cysteine. These results show that both the position of the RNA editing site and the identity of the substituted amino acid are important for channel function.     

Characterization of five RNA editing sites in Shab potassium channels

RNA editing revises the genetic code at precise locations, creating single base changes in mRNA. These changes can result in altered coding potential and modifications to protein function. Sequence analysis of the Shab potassium channel of Drosophila melanogaster revealed five such RNA editing sites. Four are constitutively edited (I583V, T643A, Y660C and I681V) and one undergoes developmentally regulated editing (T671A). These sites are located in the S4, S5-S6 loop and the S6 segments of the channel. We examined the biophysical consequences of editing at these sites by creating point mutations, each containing the genomic (unedited) base at one of the five sites in the background of a channel in which all other sites are edited. We also created a completely unedited construct. The function of these constructs was characterized using two-microelectrode voltage clamp in Xenopus oocytes. Each individual 'unediting' mutation slowed the time course of deactivation and the rise time during channel activation. Two of the mutants exhibited significant hyperpolarized shifts in their midpoints of activation. Constructs that deactivated slowly also inactivated slowly, supporting a mechanism of closed-state inactivation. One of the editing sites, position 660, aligns with the Shaker 449 residue, which is known to be important in tetraethylammonium (TEA) block. The aromatic, genomically-encoded residue tyrosine at this position in Shab enhances TEA block 14 fold compared to the edited residue, cysteine. These results show that both the position of the RNA editing site and the identity of the substituted amino acid are important for channel function.     

RNA Editing in the Central Cavity as a Mechanism to Regulate Surface Expression of the Voltage-gated Potassium Channel Kv1.1

Voltage-gated potassium (Kv) 1.1 channels undergo a specific enzymatic RNA deamination, generating a channel with a single amino acid exchange located in the inner pore cavity (Kv1.1^I400V). We studied I400V-edited Kv1.1 channels in more detail and found that Kv1.1^I400V gave rise to much smaller whole-cell currents than Kv1.1. To elucidate the mechanism behind this current reduction, we conducted electrophysiological recordings on single-channel level and did not find any differences. Next we examined channel surface expression in Xenopus oocytes and HeLa cells using a chemiluminescence assay and found the edited channels to be less readily expressed at the surface membrane. This reduction in surface expression was verified by fluorescence imaging experiments. Western blot analysis for comparison of protein abundances and glycosylation patterns did not show any difference between Kv1.1 and Kv1.1^I400V, further indicating that changed trafficking of Kv1.1^I400V is causing the current reduction. Block of endocytosis by dynasore or AP180C did not abolish the differences in current amplitudes between Kv1.1 and Kv1.1^I400V, suggesting that backward trafficking is not affected. Therefore, our data suggest that I400V RNA editing of Kv1.1 leads to a reduced current size by a decreased forward trafficking of the channel to the surface membrane. This effect is specific for Kv1.1 because coexpression of Kv1.4 channel subunits with Kv1.1^I400V abolishes these trafficking effects. Taken together, we identified RNA editing as a novel mechanism to regulate homomeric Kv1.1 channel trafficking. Fine-tuning of Kv1.1 surface expression by RNA editing might contribute to the complexity of neuronal Kv channel regulation.     

RNA editing in eag potassium channels

RNA editing at four sites in eag, a Drosophila voltage-gated potassium channel, results in the substitution of amino acids into the final protein product that are not encoded by the genome. These sites and the editing alterations introduced are K467R (Site 1, top of the S6 segment), Y548C, N567D and K699R (sites 2–4, within the cytoplasmic C-terminal domain). We mutated these residues individually and expressed the channels in Xenopus oocytes. A fully edited construct (all four sites) has the slowest activation kinetics and a paucity of inactivation, whereas the fully unedited channel exhibits the fastest activation and most dramatic inactivation. Editing Site 1 inhibits steady-state inactivation. Mutating Site 1 to the neutral residues resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship. Activation kinetics display a Cole-Moore shift that is enhanced by RNA editing. Normalized open probability relationships for 467Q, 467R and 467K are superimposable, indicating little effect of the mutations on steady-state activation. 467Q and 467R enhance instantaneous inward rectification, indicating a role of this residue in ion permeation. Intracellular tetrabutylammonium blocks 467K significantly better than 467R. Block by intracellular, but not extracellular, tetraethylammonium interferes with inactivation. The fraction of inactivated current is reduced at higher extracellular Mg⁺² concentrations, and channels edited at Site 1 are more sensitive to changes in extracellular Mg⁺² than unedited channels. These results show that even a minor change in amino acid side-chain chemistry and size can have a dramatic impact on channel biophysics, and that RNA editing is important for fine-tuning the channel’s function.     

RNA editing in eag potassium channels: Biophysical consequences of editing a conserved S6 residue

RNA editing at four sites in eag, a Drosophila voltage-gated potassium channel, results in the substitution of amino acids into the final protein product that are not encoded by the genome. These sites and the editing alterations introduced are K467R (Site 1, top of the S6 segment), Y548C, N567D and K699R (sites 2–4, within the cytoplasmic C-terminal domain). We mutated these residues individually and expressed the channels in Xenopus oocytes. A fully edited construct (all four sites) has the slowest activation kinetics and a paucity of inactivation, whereas the fully unedited channel exhibits the fastest activation and most dramatic inactivation. Editing Site 1 inhibits steady-state inactivation. Mutating Site 1 to the neutral residues resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship. Activation kinetics display a Cole-Moore shift that is enhanced by RNA editing. Normalized open probability relationships for 467Q, 467R and 467K are superimposable, indicating little effect of the mutations on steady-state activation. 467Q and 467R enhance instantaneous inward rectification, indicating a role of this residue in ion permeation. Intracellular tetrabutylammonium blocks 467K significantly better than 467R. Block by intracellular, but not extracellular, tetraethylammonium interferes with inactivation. The fraction of inactivated current is reduced at higher extracellular Mg⁺² concentrations, and channels edited at Site 1 are more sensitive to changes in extracellular Mg⁺² than unedited channels. These results show that even a minor change in amino acid side-chain chemistry and size can have a dramatic impact on channel biophysics, and that RNA editing is important for fine-tuning the channel’s function.     

Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization

We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.     

Syntaxin 1A regulates surface expression of beta-cell ATP-sensitive potassium channels

The pancreatic ATP-sensitive potassium (K(ATP)) channel consisting of four inwardly rectifying potassium channel 6.2 (Kir6.2) and four sulfonylurea receptor SUR1 subunits plays a key role in insulin secretion by linking glucose metabolism to membrane excitability. Syntaxin 1A (Syn-1A) is a plasma membrane protein important for membrane fusion during exocytosis of insulin granules. Here, we show that Syn-1A and K(ATP) channels endogenously expressed in the insulin-secreting cell INS-1 interact. Upregulation of Syn-1A by overexpression in INS-1 leads to a decrease, whereas downregulation of Syn-1A by small interfering RNA (siRNA) leads to an increase, in surface expression of K(ATP) channels. Using COSm6 cells as a heterologous expression system for mechanistic investigation, we found that Syn-1A interacts with SUR1 but not Kir6.2. Furthermore, Syn-1A decreases surface expression of K(ATP) channels via two mechanisms. One mechanism involves accelerated endocytosis of surface channels. The other involves decreased biogenesis and processing of channels in the early secretory pathway. This regulation is K(ATP) channel specific as Syn-1A has no effect on another inward rectifier potassium channel Kir3.1/3.4. Our results demonstrate that in addition to a previously documented role in modulating K(ATP) channel gating, Syn-1A also regulates K(ATP) channel expression in β-cells. We propose that physiological or pathological changes in Syn-1A expression may modulate insulin secretion by altering glucose-secretion coupling via changes in K(ATP) channel expression.     

Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin

Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.     

A novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide: protein kinase A-dependent endocytosis

The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (K(ATP)) and calcium-activated (K(CA)) potassium channels have been extensively studied, but less is known about the role of voltage-gated (K(V)) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of K(V)1.4 via activation of protein kinase A (PKA). Using mutant forms of K(V)1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant K(V)1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of K(V)1.4. Expression of K(V)1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells (clone 832/13) of wild-type (WT) K(V)1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT K(V)1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating K(V)1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.     

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.     

A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation

The inward rectifier K(+) channel Kir2.1 mediates the potassium I(K1) current in the heart. It is encoded by KCNJ2 gene that has been linked to Andersen's syndrome. Recently, strong evidences showed that Kir2.1 channels were associated with mouse atrial fibrillation (AF), therefore we hypothesized that KCNJ2 was associated with familial AF. Thirty Chinese AF kindreds were evaluated for mutations in KCNJ2 gene. A valine-to-isoleucine mutation at position 93 (V93I) of Kir2.1 was found in all affected members in one kindred. This valine and its flanking sequence is highly conserved in Kir2.1 proteins among different species. Functional analysis of the V93I mutant demonstrated a gain-of-function consequence on the Kir2.1 current. This effect is opposed to the loss-of-function effect of previously reported mutations in Andersen's syndrome. Kir2.1 V93I mutation may play a role in initiating and/or maintaining AF by increasing the activity of the inward rectifier K(+) channel.     

KCNE4 can co-associate with the I(Ks) (KCNQ1-KCNE1) channel complex

Voltage-gated potassium (K(V)) channels can form heteromultimeric complexes with a variety of accessory subunits, including KCNE proteins. Heterologous expression studies have demonstrated diverse functional effects of KCNE subunits on several K(V) channels, including KCNQ1 (K(V)7.1) that, together with KCNE1, generates the slow-delayed rectifier current (I(Ks)) important for cardiac repolarization. In particular, KCNE4 exerts a strong inhibitory effect on KCNQ1 and other K(V) channels, raising the possibility that this accessory subunit is an important potassium current modulator. A polyclonal KCNE4 antibody was developed to determine the human tissue expression pattern and to investigate the biochemical associations of this protein with KCNQ1. We found that KCNE4 is widely and variably expressed in several human tissues, with greatest abundance in brain, liver and testis. In heterologous expression experiments, immunoprecipitation followed by immunoblotting was used to establish that KCNE4 directly associates with KCNQ1, and can co-associate together with KCNE1 in the same KCNQ1 complex to form a 'triple subunit' complex (KCNE1-KCNQ1-KCNE4). We also used cell surface biotinylation to demonstrate that KCNE4 does not impair plasma membrane expression of either KCNQ1 or the triple subunit complex, indicating that biophysical mechanisms probably underlie the inhibitory effects of KCNE4. The observation that multiple KCNE proteins can co-associate with and modulate KCNQ1 channels to produce biochemically diverse channel complexes has important implications for understanding K(V) channel regulation in human physiology.     

Hepatitis delta virus minimal substrates competent for editing by ADAR1 and ADAR2

A host-mediated RNA-editing event allows hepatitis delta virus (HDV) to express two essential proteins, the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L), from a single open reading frame. One or several members of the ADAR (adenosine deaminases that act on RNA) family are thought to convert the adenosine to an inosine (I) within the HDAg-S amber codon in antigenomic RNA. As a consequence of replication, the UIG codon is converted to a UGG (tryptophan [W]) codon in the resulting HDAg-L message. Here, we used a novel reporter system to monitor the editing of the HDV amber/W site in the absence of replication. In cultured cells, we observed that both human ADAR1 (hADAR1) and hADAR2 were capable of editing the amber/W site with comparable efficiencies. We also defined the minimal HDV substrate required for hADAR1- and hADAR2-mediated editing. Only 24 nucleotides from the amber/W site were sufficient to enable efficient editing by hADAR1. Hence, the HDV amber/W site represents the smallest ADAR substrate yet identified. In contrast, the minimal substrate competent for hADAR2-mediated editing contained 66 nucleotides.     

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.