Ito channels are octomeric complexes with four subunits of each Kv4.2 and K+ channel-interacting protein 2

Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits.     

Plant K+ channel alpha-subunits assemble indiscriminately.

In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

The sigma receptor as a ligand-regulated auxiliary potassium channel subunit

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.     

Palmitoylation of KChIP splicing variants is required for efficient cell surface expression of Kv4.3 channels

The Ca(2+)-binding proteins KChIP1-4 (KChIP3 is also known as DREAM and calsenilin) act as auxiliary subunits for voltage-gated K(+) channels in the Kv4 family. Here we identify three splicing isoforms of rat KChIP2 with variable N-terminal peptides. The two longer isoforms, which contain the 32-amino acid peptide, produce larger increases in Kv4.3 protein level and current density and more effectively localize themselves and their associated channels at the plasma membrane than the shortest variant. The 32-amino acid peptide contains potential palmitoylation cysteines. Metabolic labeling demonstrates that these cysteines in the KChIP2 isoforms, as well as the corresponding sites in KChIP3, are palmitoylated. Mutating these cysteines reduces their plasma membrane localization and the enhancement of Kv4.3 current density. Thus, palmitoylation of the KChIP auxiliary subunits controls plasma membrane localization of their associated channels.     

The bacterial K+ channel structure and its implications for neuronal channels.

Voltage-gated K+ (Kv) channels play a central role in generating action potentials and rhythmic patterns, as well as in dendritic signal processing in neurons. Recently, the first structure of a member of the K+ channel family was solved. Although this channel is from bacteria and has a streamlined body plan with no voltage gating, it establishes the architecture of the functional core of the voltage-gated (K+) channels and their relatives. This architecture explains the crucial features of ion permeation and blockade, and gives some strong hints about gating. The bacterial K+ channel structure is the central piece in a puzzle; it remains to be seen how it will fit together with other domains of the Kv channels, with auxiliary subunits, and with other signal transduction molecules.     

G protein beta2 subunit-derived peptides for inhibition and induction of G protein pathways. Examination of voltage-gated Ca2+ and G protein inwardly rectifying K+ channels

Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K+ channels (GIRK) are modulated via direct binding of G proteins. The modulation is mediated by G protein betagamma subunits. By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the modulatory domains of the G protein beta subunit on the recombinant P/Q-type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2+ currents of cultured hippocampal neurons. We found that Gbeta2 subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein modulation of the examined ion channels. In particular, the 25-amino acid peptide derived from the Gbeta2 N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the Gbeta2 C terminus induced modulation of voltage-gated Ca2+ channels and GIRK channels. Fluorescence resonance energy transfer (FRET) analysis of the live action of these peptides revealed that the 25-amino acid peptide diminished the FRET signal between G protein beta2gamma3 subunits, indicating a reorientation between G protein beta2gamma3 subunits in the presence of the peptide. In contrast, the 35-amino acid peptide increased the FRET signal between GIRK1,2 channel subunits, similarly to the Gbetagamma-mediated FRET increase observed for this GIRK subunit combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is structured. These results indicate that individual G protein beta subunit domains can act as independent, separate modulatory domains to either induce or inhibit G protein modulation for several effector proteins.     

Modulation of Kv4-encoded K(+) currents in the mammalian myocardium by neuronal calcium sensor-1

Voltage-gated K(+) channels are multimeric proteins, consisting of four pore-forming alpha-subunits alone or in association with accessory subunits. Recently, for example, it was shown that the accessory Kv channel interacting proteins form complexes with Kv4 alpha-subunits and modulate Kv4 channel activity. The experiments reported here demonstrate that the neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily, is expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from (adult mouse) ventricular extracts. In addition, co-expression studies in HEK-293 cells reveal that NCS-1 increases membrane expression of Kv4 alpha-subunits and functional Kv4-encoded K(+) current densities. Co-expression of NCS-1 also decreases the rate of inactivation of Kv4 alpha-subunit-encoded K(+) currents. In contrast to the pronounced effects of Kv channel interacting proteins on Kv4 channel gating, however, NCS-1 co-expression does not measurably affect the voltage dependence of steady-state inactivation or the rate of recovery from inactivation of Kv4-encoded K(+) currents. Taken together, these results suggest that NCS-1 is an accessory subunit of Kv4-encoded I(to,f) channels that functions to regulate I(to,f) density in the mammalian myocardium.     

Coupling between voltage sensors and activation gate in voltage-gated K+ channels

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1-S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5-S6, equivalent to M1-M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4-S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.     

Voltage‐dependent κ‐opioid modulation of action potential waveform‐elicited calcium currents in neurohypophysial terminals

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca²⁺ channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca²⁺ channel kinetics, and the associated Ca²⁺ influx, is primarily controlled by membrane voltage and can be modulated, in a voltage‐dependent manner, by G‐protein subunits interacting with voltage‐gated calcium channels (VGCCs). In this series of experiments we test whether the κ‐ and µ‐opioid inhibition of Ca²⁺ currents in NH terminals is voltage‐dependent. Voltage‐dependent relief of G‐protein inhibition of VGCC can be achieved with either a depolarizing square pre‐pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the κ‐ and µ‐receptors in neurohypophysial terminals. The κ‐opioid VGCC inhibition is relieved by such pre‐pulses, suggesting that this receptor is involved in a voltage‐dependent membrane delimited pathway. In contrast, µ‐opioid inhibition of VGCC is not relieved by such pre‐pulses, indicating a voltage‐independent diffusible second‐messenger signaling pathway. Furthermore, relief of κ‐opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity‐dependent modulation in vivo. Differences in the facilitation of Ca²⁺ channels due to specific G‐protein modulation during a burst of APs may contribute to the fine‐tuning of Ca²⁺‐dependent neuropeptide release in other CNS terminals, as well. J. Cell. Physiol. 225: 223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Structure of a voltage-dependent K+ channel beta subunit.

The integral membrane subunits of many voltage-dependent potassium channels are associated with an additional protein known as the beta subunit. One function of beta subunits is to modify K+ channel gating. We have determined the structure of the conserved core of mammalian beta subunits by X-ray crystallography at 2.8 A resolution. Like the integral membrane component of K+ channels, beta subunits form a four-fold symmetric structure. Each subunit is an oxidoreductase enzyme complete with a nicotinamide co-factor in its active site. Several structural features of the enzyme active site, including its location with respect to the four-fold axis, imply that it may interact directly or indirectly with the K+ channel's voltage sensor. This structure suggests a mechanism for coupling membrane electrical excitability directly to chemistry of the cell.     

Steady-state and closed-state inactivation properties of inactivating BK channels

Calcium-dependent potassium (BK-type) Ca2+ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 alpha subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca2+] from 1 to 60 microM. At 10 and 60 microM Ca2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca2+-dependent inactivation. In contrast, in the absence of Ca2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.     

Identification of proteins resembling G-protein alpha subunits in locust muscle

In locust skeletal muscle, FMRFamide-like peptides decrease a K+ conductance. Functional data suggest the involvement of G-proteins. For identification of G-protein alpha-subunits, membranes of locust skeletal muscle were probed with ADP-ribosylating bacterial toxins, the photoreactive GTP analog, [alpha-32P]GTP azidoanilide, and with antibodies against mammalian alpha-subunits. Multiple guanine nucleotide-binding proteins of approximately 24-95 kDa were detected. Pertussis toxin catalyzed the ADP-ribosylation of two proteins comigrating with the ADP-ribosylated alpha-subunits of the mammalian G-proteins Go and Gi. Cholera toxin promoted ADP-ribosylation of a protein comigrating with mammalian cholera toxin substrates (i.e., Gs alpha-subunits). An antibody against mammalian Go alpha-subunits detected a 54-kDa protein. Thus proteins with properties of mammalian G-protein subunits are present in insect muscle.     

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.     

A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids.

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.     

Colloquium 9: Structural and Molecular Dissection of the Node of Ranvier. Sodium channel β subunits: multifunctional molecules at the node of Ranvier

Voltage‐gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore‐forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage‐gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore‐forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin.     

14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells

Outward-rectifying K+ channels are modulated in response to environmental stimuli by a range of intracellular factors, such as cytoplasmic Ca2+, pH, kinases and phosphatases. Here we report that voltage-dependent outward-rectifying K+ channels in tomato cells are also targets for modulation by 14-3-3 proteins. In whole-cell patch-clamp experiments, recombinant 14-3-3 protein (tomato isoform TFT7) was introduced into tomato cell protoplasts via the patch pipette. As a result the steady-state outward K+ current increased twofold and this increase was not dependent upon the presence of cytoplasmic ATP. A phosphorylated peptide that contained a phosphorylated 14-3-3 target-binding motif (RSTS*TP), derived from nitrate reductase, blocked the effect of 14-3-3, thus showing the specific nature of 14-3-3 action. Kinetic parameters of the conductance, like (de)activation kinetics, voltage dependence of gating and activation potential, were not significantly different between control and 14-3-3 infused cells. Analysis of single-channel activity and whole-cell noise indicated that the single-channel conductance was not affected by 14-3-3 infusion. We conclude that 14-3-3 proteins recruit 'sleepy' channels into a voltage-activatable state. The molecular mechanism underlying the 1 : 1 ratio of constitutively active and 14-3-3 recruited channels is discussed in the light of known functions of 14-3-3 dimers.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

A BK (Slo1) channel journey from molecule to physiology

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca²⁺ and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca²⁺ sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.     

A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly

The LQT1 locus (KCNQ1) has been correlated with the most common form of inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and high risk of sudden death. The KCNQ1 gene encodes KvLQT1 alpha-subunits, which together with auxiliary IsK (KCNE1, minK) subunits form IK(s) K(+) channels. Mutant KvLQT1 subunits may be associated either with an autosomal dominant form of inherited LQT, Romano-Ward syndrome, or an autosomal recessive form, Jervell and Lange-Nielsen syndrome (JLNS). We have identified a small domain between residues 589 and 620 in the KvLQT1 C-terminus, which may function as an assembly domain for KvLQT1 subunits. KvLQT1 C-termini do not assemble and KvLQT1 subunits do not express functional K(+) channels without this domain. We showed that a JLN deletion-insertion mutation at KvLQT1 residue 544 eliminates important parts of the C-terminal assembly domain. Therefore, JLN mutants may be defective in KvLQT1 subunit assembly. The results provide a molecular basis for the clinical observation that heterozygous JLN carriers show slight cardiac dysfunctions and that the severe JLNS phenotype is characterized by the absence of KvLQT1 channel.