Functional and clinical characterization of a mutation in KCNJ2 associated with Andersen-Tawil syndrome.

BACKGROUND: Andersen-Tawil syndrome (ATS) is a rare inherited disorder, characterised by periodic paralysis, cardiac dysarrhythmias, and dysmorphic features, and is caused by mutations in the gene KCNJ2, which encodes the inward rectifier potassium channel, Kir2.1. This study sought to analyse KCNJ2 in patients with familial ATS and to determine the functional characteristics of the mutated gene. METHODS AND RESULTS: We screened a family with inherited ATS for the mutation in KCNJ2, using direct DNA sequencing. A missense mutation (T75R) of Kir2.1, located in the highly conserved cytoplasmic N-terminal domain, was identified in three affected members of this family. Using the Xenopus oocyte expression system and whole cell voltage clamp analyses, we found that the T75R mutant was non-functional and possessed a strong dominant negative effect when co-expressed with the same amount of wild type Kir2.1. Transgenic (Tg) mice expressing the mutated form of Kir2.1 in the heart had prolonged QTc intervals compared with mice expressing the wild type protein. Ventricular tachyarrhythmias were observed in 5 of 14 T75R-Tg mice compared with 1 of 7 Wt-Tg and none of 6 non-transgenic littermates. In three of five T75R-Tg mice with ventricular tachycardia, their ECG disclosed bidirectional tachycardia as in our proband. CONCLUSIONS: The in vitro studies revealed that the T75R mutant of Kir2.1 had a strong dominant negative effect in the Xenopus oocyte expression system. It still preserved the ability to co-assemble and traffic to the cell membrane in mammalian cells. For in vivo studies, the T75R-Tg mice had bidirectional ventricular tachycardia after induction and longer QT intervals.

A central role for the T1 domain in voltage-gated potassium channel formation and function.

To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the full-length functional channel.

The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK.

The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type potassium channel that passes an A-type current and is localized to dendrites and cell bodies in the hippocampus. The sequence of Kv4.2 contains several consensus sites for ERK phosphorylation. In the present studies, we tested the hypothesis that Kv4.2 is an ERK substrate. We determined that the Kv4.2 C-terminal cytoplasmic domain is an effective ERK2 substrate, and that it is phosphorylated at three sites: Thr(602), Thr(607), and Ser(616). We used this information to develop antibodies that recognize Kv4.2 phosphorylated by ERK2. One of our phospho-site-selective antibodies was generated using a triply phosphorylated peptide as the antigen. We determined that this antibody recognizes ERK-phosphorylated Kv4.2 in COS-7 cells transfected with Kv4.2 and native ERK-phosphorylated Kv4.2 in the rat hippocampus. These observations indicate that Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERK may regulate potassium-channel function by direct phosphorylation of the pore-forming alpha subunit.

Voltage dependent activation of potassium channels is coupled to T1 domain structure.

The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.

Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase.

Recent evidence suggests that K(+) channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myocytes, and activation of the cAMP second messenger cascade has been shown to modulate this transient current. We determined if Kv4.2 alpha-subunits were directly phosphorylated by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that both of these fusion proteins were substrates for PKA in vitro. By using phosphopeptide mapping and amino acid sequencing, we identified PKA phosphorylation sites on the amino- and carboxyl-terminal fusion proteins corresponding to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate PKA site phosphorylation in situ, phospho-selective antisera for each of the sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed modulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal area CA1. These results provide insight into likely sites of regulation of Kv4.2 by PKA.

Towards the three-dimensional structure of voltage-gated potassium channels.

Electrical excitability is a fundamental property of the neuromuscular systems of metazoans. The varied response of neurons to electrical excitation is largely accounted for by a diverse set of voltage-gated potassium (KV) channels in the excitable membrane. The complete structure of a KV channel is not yet available. However, recent structural biological experiments have begun to provide new insight into how specific KV channels are formed and regulated, and how they function and interact with other proteins. In particular, the selectivity of KV channels for K+ and suggestions as to how these structural elements might assemble into a functional KV channel are discussed.

Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression.

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+ channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+ channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+ channel gene. Paradoxically, we find that addition of the extra K+ channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+ channel gene dosage leads to dysregulation of normal K+ channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.

Zn2+-binding and molecular determinants of tetramerization in voltage-gated K+ channels.

The N-terminal, cytoplasmic tetramerization domain (T1) of voltage-gated K+ channels encodes molecular determinants for subfamily-specific assembly of alpha-subunits into functional tetrameric channels. Crystal structures of T1 tetramers from Shaw and Shaker subfamilies reveal a common four-layered scaffolding. Within layer 4, on the hypothetical membrane-facing side of the tetramer, the Shaw T1 tetramer contains four zinc ions; each is coordinated by a histidine and two cysteines from one monomer and by one cysteine from an adjacent monomer. The amino acids involved in coordinating the Zn2+ ion occur in a HX5CX20CC sequence motif that is highly conserved among all Shab, Shaw and Shal subfamily members, but is not found in Shaker subfamily members. We demonstrate by coimmunoprecipitation that a few characteristic residues in the subunit interface are crucial for subfamily-specific tetramerization of the T1 domains.

Crystal structure of the tetramerization domain of the Shaker potassium channel.

Voltage-dependent, ion-selective channels such as Na+, Ca2+ and K+ channel proteins function as tetrameric assemblies of identical or similar subunits. The clustering of four subunits is thought to create an aqueous pore centred at the four-fold symmetry axis. The highly conserved, amino-terminal cytoplasmic domain (approximately 130 amino acids) immediately preceding the first putative transmembrane helix S1 is designated T1. It is known to confer specificity for tetramer formation, so the heteromeric assembly of K+-channel subunits is an important mechanism for the observed channel diversity. We have determined the crystal structure of the T1 domain of a Shaker potassium channel at 1.55 A resolution. The structure reveals that four identical subunits are arranged in a four-fold symmetry surrounding a centrally located pore about 20 A in length. Subfamily-specific assembly is provided primarily by polar interactions encoded in a conserved set of amino acids at its tetramerization interface. Most highly conserved amino acids in the T1 domain of all known potassium channels are found in the core of the protein, indicating a common structural framework for the tetramer assembly.

Shaker K+ channel T1 domain self-tetramerizes to a stable structure.

The potassium channel T1 domain plays an important role in the regulated assembly of subunit proteins. We have examined the assembly properties of the Shaker channel T1 domain to determine if the domain can self-assemble, the number of subunits in a multimer, Ns and the mechanism of assembly. High pressure liquid chromatography (HPLC) size exclusion chromotography (SEC) separates T1 domain proteins into two peaks. By co-assembly assays, these peaks are identified to be a high molecular weight assembled form and a low molecular weight monomeric form. To determine the Ns of the assembled protein peak on HPLC SEC, we first cross-linked the T1 domain proteins and then separated them on HPLC. Four evenly spaced bands co-migrate with the assembled protein peak; thus, the T1 domain assembles to form a tetramer. The absence of separate dimeric and trimeric peaks of assembled T1 domain protein suggests that the tetramer is the stable assembled state, most probably a closed ring structure.

Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K+ channel subunit proteins.

We are analyzing features of the K+ channel subunit proteins that are critical for function and regulation of these proteins. Our studies show biochemically that subunit proteins from the Shaker and Shaw subfamilies fail to assemble into a heteromultimer. The basis for this incompatibility is the sequences contained within the T1 assembly domain. For a subunit protein to heteromultimerize with a Shaker subunit protein, two regions within the T1 domain, A and B, must be of the Shaker subtype. Finally, we show that the incompatibility of a Shaw A region for assembly with a Shaker protein depends upon the composition of a 30 amino acid conserved sequence in the A region.

Deletion analysis of K+ channel assembly.

An understanding of K+ channel structure is a critical step in developing an appreciation of the function and regulation of these proteins. We have begun a biochemical analysis of the early steps in K+ channel formation following translation into endoplasmic reticulum membranes. In our experiments, a series of K+ channel subunit protein deletions were constructed and then tested for posttranslational processing and assembly. We find that all deletions containing the S1 domain are inserted into the membrane. The loop between S1 and S2 is glycosylated; thus, this segment is topologically extracellular. Translated subunit proteins mix in the membrane, then assemble into tetramers. This subunit assembly is critically driven by a conserved, self-tetramerizing sequence in the N-terminal cytoplasmic region, which we have named the tetramerization 1 domain.

Overexpression of an Aplysia shaker K+ channel gene modifies the electrical properties and synaptic efficacy of identified Aplysia neurons.

Although potassium channels play a variety of roles in shaping the electrical properties of neurons, little is known about how these channels are constituted in neurons. To examine the assembly and physiological function of A-type K+ channels in mature differentiated neurons, we have developed a highly efficient gene transfer method for Aplysia neurons that has allowed us to express about 10(7) copies of the cloned Aplysia Shaker (Sh) K+ channel (AK01a) in single identified cells. We find that expression of AK01a phenocopies one of the native transient K+ currents (IAdepol), suggesting that the native channel carrying IAdepol is assembled as a homooligomer of AK01a. Overexpression of AK01a has substantial effect on the action potential, shortening its duration, enhancing its hyperpolarizing afterpotential, and depressing by more than half the amount of transmitter release by the action potential from the terminals. Thus, the AK01a channel not only contributes to the firing properties within a given neuron but also can regulate the signaling between interconnected cells.

Cloning and expression of an Aplysia K+ channel and comparison with native Aplysia K+ currents.

We describe here the cloning of the Aplysia K+ channel AK01a.AK01a codes for a protein of 515 amino acids, shows considerable homology to other cloned potassium channels, and can be classified as a member of the ShakerK+ channel family. Expression of the AK01a channel in Xenopus oocytes produces a rapidly inactivating outward potassium current (IAK01a) resembling the A-type currents of Drosophila Shaker. Gating for this current is shifted to potentials considerably more positive than the traditional A-currents of Aplysia; we have, however, identified a novel transient potassium current (IAdepoI) in a subset of Aplysia neurons that has similar gating and pharmacological properties to IAK01a.

Agonists that suppress M-current elicit phosphoinositide turnover and Ca2+ transients, but these events do not explain M-current suppression.

The hypothesis that acetylcholine, substance P, and LHRH suppress M-current by activating phospholipase C was tested. Each agonist caused turnover of phosphoinositide, as measured by release of inositol phosphates, and a modest transient rise in intracellular free Ca2+ ([ Ca2+]i), as determined with fura-2. Active phorbol esters depressed M-current only 50% and did not prevent further suppression by LHRH. M-current, its control by agonists, and its depression by phorbol esters were not affected by adding inositol trisphosphate or Ca2+ buffers with high or low Ca2+ to the whole-cell, voltage-clamp pipette. We conclude that phospholipase C activation does occur but does not mediate the suppression of M-current by agonists. Caffeine produced large [Ca2+]i transients and acted as an agonist to suppress M-current.

GTP-binding proteins couple cardiac muscarinic receptors to a K channel.

Binding of acetylcholine (ACh) to cardiac muscarinic ACh receptors (mAChR) activates a potassium channel that slows pacemaker activity. Although the time course of this activation suggests a multi-step process with intrinsic delays of 30-100 ms, no second-messenger system has been demonstrated to link the mAChR to the channel. Changes in cyclic nucleotide levels (cyclic AMP and cyclic GMP) do not affect this K channel or its response to muscarinic agonists. Indeed, electrophysiological experiments argue against the involvement of any second messenger that diffuses through the cytoplasm. We report here that coupling of the mAChR in embryonic chick atrial cells to this inward rectifying K channel requires intracellular GTP. Furthermore, pretreatment of cells with IAP (islet-activating protein from the bacterium Bordetella pertussis) eliminates the ACh-induced inward rectification. As IAP specifically ADP-ribosylates two GTP-binding proteins, Ni and No, that can interact with mAChRs, we conclude that a guanyl nucleotide-binding protein couples ACh binding to channel activation. This represents the first demonstration that a GTP-binding protein can regulate the function of an ionic channel without acting through cyclic nucleotide second messengers.