Publications
2024
Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 926 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.
2023
Store-operated calcium entry (SOCE) is a vital process aimed at refilling cellular internal Ca2+ stores and a primary cellular signaling driver for transcription factors entry to the nucleus. SOCE-associated regulatory factor (SARAF)/TMEM66 is an endoplasmic reticulum (ER)-resident transmembrane protein that promotes SOCE inactivation and prevents Ca2+ overfilling of the cell. Here, we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass, and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamuspituitaryadrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the hypothalamus's paraventricular nucleus (PVN) neurons reduces old age-induced obesity and preserves locomotor activity, lean mass, and energy expenditure, suggesting a possible central control with a site-specific role for SARAF. At the cellular level, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca2+ oscillations, and an increased mitochondrial spare respiratory capacity (SPC), thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses.
2022
Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.
2021
Many venomous organisms carry in their arsenal short polypeptides that block K+ channels in a highly selective manner. These toxins may compete with the permeating ions directly via a \u201cplug\u201d mechanism or indirectly via a \u201cpore-collapse\u201d mechanism. An alternative \u201clid\u201d mechanism was proposed but remained poorly defined. Here we study the Drosophila Shaker channel block by Conkunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom. Despite their similarity, the two peptides exhibited differences in their binding poses and biophysical assays, implying discrete action modes. We show that while Conkunitzin-S1 binds tightly to the channel turret and acts via a \u201cpore-collapse\u201d mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism we term a \u201cmolecular-lid\u201d. Our study provides an atomic description of the \u201clid\u201d K+ blocking mode and offers valuable insights for the design of therapeutics based on venom peptides.
It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.
Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzymemediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QMbased inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QMbased inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolatetoQM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cisacting QMbased substrates could be used as activitybased probes to identify various phospho and phosphonoester hydrolases, and potentially other hydrolases. Internal affairs: Phosphoester substrates that release electrophilic quinonemethides were applied as activitybased probes in three phosphotriesterases that differ in mechanism and level of activity. Their substrates reactivity, and mode of labeling, were systematically investigated with the aim of achieving specific cis labeling rather than trans.
GIRK channels are essential for the slow inhibition of electrical activity in the nervous system and heart rate regulation via the parasympathetic system. The implications of individual GIRK isoforms in specific physiological activities are based primarily on studies conducted with GIRKnull mouse lines. Here we utilize a novel knockin mouse line in which YFP was fused inframe to the Nterminus of GIRK1 (YFPGIRK1) to correlate GIRK1 spatial distribution with physiological activities. These mice, however, displayed spontaneous seizurelike activity and thus were investigated for the origin of such activity. We show that GIRK tetramers containing YFPGIRK1 are correctly assembled and trafficked to the plasma membrane, but are functionally impaired. A battery of behavioural assays conducted on YFPGIRK1 and GIRK1null (GIRK1−/−) mice revealed similar phenotypes, including impaired nociception, reduced anxiety and hyperactivity in an unfamiliar environment. However, YFPGIRK1 mice exhibited increased homecage locomotion while GIRK1−/− mice did not. In addition, we show that the GIRK1 subunit is essential for intact spatial learning and memory and synaptic plasticity in hippocampal brain slices. This study expands our knowledge regarding the role of GIRK1 in neuronal processes and underlines the importance of GIRK1containing heterotetramers.
Animal venom is a rich source for peptide toxins that bind and modulate the function of ion channels. Owing to their ability to bind receptor sites on the channel protein with high affinity and specificity, peptide neurotoxins have become an indispensable tool for ion channel research. Recent breakthroughs in structural biology and advances in computer simulations of biomolecules have sparked a new interest in animal toxins as probes of channel protein structure and function. Here, we focus on methods used to produce animal toxins for research purposes using recombinant expression. The specific challenges associated with heterologous production of venom peptides are discussed, and several methods targeting these issues are presented with an emphasis on E. coli based systems. An efficient protocol for the bacterial expression, folding, and purification of recombinant venom peptides is described.
Ion channels can tailor their activity to the particular cellular context by incorporating auxiliary subunits that are channel-type specific. In this issue of Cell, Avalos Prado et al. now find that a well-characterized voltage-gated K+ channel auxiliary subunit can also modulate the gating of Ca2+-activated Cl- channels.
2019
Background and Purpose A second-generation antihistamine, terfenadine, is known to induce arrhythmia by blocking hERG channels. In this study, we have shown that terfenadine also inhibits the activity of G-protein-gated inwardly rectifying K+ (GIRK) channels, which regulate the excitability of neurons and cardiomyocytes. To clarify the underlying mechanism(s), we examined the effects of several antihistamines on GIRK channels and identified the structural determinant for the inhibition. Experimental Approach Electrophysiological recordings were made in Xenopus oocytes and rat atrial myocytes to analyse the effects of antihistamines on various GIRK subunits (K(ir)3.x). Mutagenesis analyses identified the residues critical for inhibition by terfenadine and the regulation of ion selectivity. The potential docking site of terfenadine was analysed by molecular docking. Key Results GIRK channels containing K(ir)3.1 subunits heterologously expressed in oocytes and native GIRK channels in atrial myocytes were inhibited by terfenadine and other non-sedating antihistamines. In K(ir)3.1 subunits, mutation of Phe137, located in the centre of the pore helix, to the corresponding Ser in K(ir)3.2 subunits reduced the inhibition by terfenadine. Introduction of an amino acid with a large side chain in K(ir)3.2 subunits at Ser148 increased the inhibition. When this residue was mutated to a non-polar amino acid, the channel became permeable to Na+. Phosphoinositide-mediated activity was also decreased by terfenadine. Conclusion and Implications The Phe137 residue in K(ir)3.1 subunits is critical for inhibition by terfenadine. This study provides novel insights into the regulation of GIRK channels by the pore helix and information for drug design.
Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of K(v)1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.
Voltage sensing by ion channels is the key event enabling the generation and propagation of electrical activity in excitable cells. In this issue of Cell, Wisedchaisri et al. provide a structural view of a voltage-gated sodium channel in its resting closed conformation.
It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.
Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the Xray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the \u201cSARAF-fold.\u201d The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the \u201cSARAF luminal switch\u201d that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation.
2018
Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons. Panayotis et al. found decreased anxiety in importin α5 knockout mice. They report that importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.
2016
Extending their accepted role in downregulating GPCRs from the cell membrane following GPCR activation, GRK shows an additional novel role, to rapidly control GPCRs activation of effectors that depend on the G protein βγ subunits (Gβγ), independent of their catalytic activity. GPCR-coupled potassium channels (GIRK) are found in excitable tissues such as neurons, heart, and endocrine organs, where they are known to decrease cells excitability following their activation by Gi/o-coupled GPCRs. In these tissues, GIRK participate in signaling systems that demand a precise temporal control, such as the regulation of heart rate and synaptic activity. While GPCRs activation can be prolonged by agonists, a constrained temporal response of GIRK channel activity can be achieved by GRKs capable of binding Gβγ subunit (GRK2 and 3). Simultaneously with GPCR activation, GRK2 binds the free Gβγ subunits through its pleckstrin homology domain immediately ceasing GIRK channel activity, in a process of fast desensitization. GIRK fast desensitization occurs with the mass action of cytosolic GRK2 recruited to the cell membrane upon receptor activation which appears simultaneously with channel current desensitization. Interestingly, GRK-mediated desensitization of GIRK currents is mediated by many but not all different Gi/o-linked GPCRs. The question whether a GPCR-mediated GRK fast desensitization relies on a specific Gβγ subunits pair coupled to a specific receptor, or on a direct precou-pling of GRK to a specific subset of the GPCRs is still an open question.
2014
Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that infl uence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems.
2013
2012
Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca2+ stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Using heterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca 2+-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca2+ signals and determining the content of the major intracellular Ca2+ stores, a role that is likely to be important in protecting cells from Ca2+ overfilling.
G protein-activated inwardly rectifying K+ channels (GIRK) generate slow inhibitory postsynaptic potentials in the brain via Gi/o protein-coupled receptors. GIRK2, a GIRK subunit, is widely abundant in the brain and has been implicated in various functions and pathologies, such as learning and memory, reward, motor coordination, and Down syndrome. Down syndrome, the most prevalent cause of mental retardation, results from the presence of an extra maternal chromosome 21 (trisomy 21), which comprises the Kcnj6 gene (GIRK2). The present study examined the behaviors and cellular physiology properties in mice harboring a single trisomy of the Kcnj6 gene. Kcnj6 triploid mice exhibit deficits in hippocampal-dependent learning and memory, altered responses to rewards, hampered depotentiation, a form of excitatory synaptic plasticity, and have accentuated long-term synaptic depression. Collectively the findings suggest that triplication of Kcnj6 gene may play an active role in some of the abnormal neurological phenotypes found in Down syndrome.
2010
G protein-coupled receptors (GPCRs) respond to agonists to activate downstream enzymatic pathways or to gate ion channel function. Turning off GPCR signaling is known to involve phosphorylation of the GPCR by GPCR kinases (GRKs) to initiate their internalization. The process, however, is relatively slow and cannot account for the faster desensitization responses required to regulate channel gating. Here, we show that GRKs enable rapid desensitization of the G protein-coupled potassium channel (GIRK/Kir3.x) through a mechanism independent of their kinase activity. On GPCR activation, GRKs translocate to the membrane and quench channel activation by competitively binding and titrating G protein bg subunits away from the channel. Of interest, the ability of GRKs to effect this rapid desensitization depends on the receptor type. The findings thus reveal a stimulus-specific, phosphorylationindependent mechanism for rapidly downregulating GPCR activity at the effector level.
2009
The traditional view of G protein-coupled receptor (GPCR)-mediated signalling puts the players in this signalling cascade, namely the GPCR, the G protein and its effector, as individual components in space, where the signalling specificity is obtained mainly by the interaction of the GPCR and the Gα subunits of the G protein. A question is then raised as to how fidelity in receptor signalling is achieved, given that many systems use the same components of the G protein signalling machinery. One possible mechanism for obtaining the specific flow of the downstream signals, from the activated G protein to its specific effector target, in a timely manner, is compartmentalization, a spatial arrangement of the complex in a rather restricted space. Here we review our recent findings related to these issues, using the G protein-coupled potassium channel (GIRK) as a model effector and fluorescence-based approaches to reveal how the signalling complex is arranged and how the G protein exerts its action to activate the GIRK channel in intact cells.
G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.
2008
Insulin secretion inhibitors (ISI) such as adrenaline and somatostatin act on the pancreatic β-cell by a number of mechanisms, one of which is plasma membrane hyperpolarization. Despite the ample evidence for this effect, the principal underlying channels have not been identified thus far. The G protein-gated inwardly rectifying potassium (Kir3.x/GIRK) channels, which are responsible for hyperpolarization in other excitable tissues, are likely candidates. In this paper, we show that GIRK channels are expressed and functional in mouse pancreatic islet cells. Reverse transcription polymerase chain reaction analysis revealed all four GIRK gene products in islet tissue. Immunofluorescent labeling of pancreatic sections demonstrated exclusive islet localization of all GIRK subunits, in part within insulin-expressing cells. Using the whole-cell configuration of the patch clamp technique, we found that the application of tertiapin-Q, a selective inhibitor of the GIRK channels, abolishes adrenaline-mediated inward currents and strongly attenuates adrenaline-induced hyperpolarization in a reversible manner. These results imply that GIRK channels are responsible for a major part of the electrical response to adrenaline in islet cells and suggest a role for these channels in pancreatic physiology.
while X-ray crystallography provides extremely high-resolution snapshot of protein structure, it lacks the ability to provide dynamic information on the processes involving conformational rearrangements of the protein. Methods to record protein conformational dynamics are present, in particular those that are based on fluorescence measurements, and are now more and more utilized in studying proteins in their natural environment. Here we describe the use of fluorescence resonance energy transfer (FRET) technique to monitor the conformational rearrangements associated with the gating of the G protein-coupled potassium channel (GIRK/Kir3.x), and its relation with the G protein subunits. The FRET technique is combined with total internal fluorescence (TIRF) microscopy, and allows the dissection of the signal originating from channel proteins that reside exclusively in the plasma membrane. Since most of the components associated with GIRK channel gating are intracellular, that involve various biochemical steps, proteins were labeled with genetically encoded variants of the green fluorescence protein and signals were acquired from live cells in culture. Using these methodologies we were able to show that gating conformational rearrangements, i.e. the opening of the channel, involve the rotation and expansion of the channel subunits cytosolic termini, along the channel's central axis. In addition, the G proteins that trigger this process reside very close to the channel, to ensure high signaling specificity and to provide temporal precision of the gating process.
2007
The function of inwardly rectifying K+ (Kir) channels is highly diverse and therefore is tightly regulated by various environmental factors. In their article in this issue of Neuron, Rapedius et al. recognize a conserved structural mechanism for Kir channels gating by both pH and PIP2. In light of these findings and accumulated knowledge, PIP2 is suggested to have a common coregulatory role in the gating of Kir channels by all their soluble modulators.
2006
G protein-coupled signaling is one of the major mechanisms for controlling cellular excitability. One of the main targets for this control at postsynaptic membranes is the G protein-coupled potassium channels (GIRK/Kir3), which generate slow inhibitory postsynaptic potentials following the activation of Pertussis toxin-sensitive G protein-coupled receptors. Using total internal reflection fluorescence (TIRF) microscopy combined with fluorescence resonance energy transfer (FRET), in intact cells, we provide evidence for the existence of a trimeric G protein-channel complex at rest. We show that activation of the channel via the receptor induces a local conformational switch of the G protein to induce channel opening. The presence of such a complex thus provides the means for a precise temporal and highly selective activation of the channel, which is required for fine tuning of neuronal excitability.
G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). Here we report that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, we further show an essential role of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability.
2005
Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.
2003
Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K+ ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.
G protein-coupled inwardly rectifying potassium channels (GIRK/Kir3) are important elements in controlling cellular excitability. In recent years, tremendous progress has been made toward understanding various components involved in channel activation, modulation, and signaling specificity. In this review, we summarize these recent findings and attempt to put them in context with recently available structural data.
G protein-coupled potassium channels (GIRK/Kir3.x) are key determinants that translate inhibitory chemical neurotransmission into changes in cellular excitability. To understand the mechanism of channel activation by G proteins, it is necessary to define the structural rearrangements in the channel that result from interaction with Gβγ subunits. In this study we used a combination of fluorescence spectroscopy and through-the-objective total internal reflection microscopy to monitor the conformational rearrangements associated with the activation of GIRK channels in single intact cells. We detect activation-induced changes in FRET consistent with a rotation and expansion of the termini along the central axis of the channel. We propose that this rotation and expansion of the termini drives the channel to open by bending and possibly rotating the second transmembrane segment.
2002
G protein coupled inwardly rectifying K+ channels (GIRK/Kir3.x) are mainly activated by a direct interaction with Gβγ subunits, released upon the activation of inhibitory neurotransmitter receptors. Although Gβγ binding domains on all four subunits have been found, the relative contribution of each of these binding sites to channel gating has not yet been defined. It is also not known whether GIRK channels open once all Gβγ sites are occupied, or whether gating is a graded process. We used a tandem tetrameric approach to enable the selective elimination of specific Gβγ binding domains in the tetrameric context. Here, we show that tandem tetramers are fully operational. Tetramers with only one wild-type channel subunit showed receptor-independent high constitutive activity. The presence of two or three wild-type subunits reconstituted receptor activation gradually. Furthermore, a tetramer with no GIRK1 Gβγ binding domain displayed slower kinetics of activation. The slowdown in activation was found to be independent of regulator of G protein signaling or receptor coupling, but this slowdown could be reversed once only one Gβγ binding domain of GIRK1 was added. These results suggest that partial activation can occur under low Gβγ occupancy and that full activation can be accomplished by the interaction with three Gβγ binding subunits.
Voltage-gated ion channels open in response to a change in membrane potential. The "sensor," or the channel's molecular entity responsible for the detection of voltage change, is formed by a transmembrane element, rich with basic residues, called the "voltage sensor" or the "S4 domain." The movement of the S4 drives a global conformational change leading to the opening of the permeation pathway and ion conduction. In this issue of Neuron, Schönherr and colleagues (2002) show that physical constrains of the "gating canal," or the crevice through which the S4 moves, determines whether voltage-gated potassium channels open quickly or slowly.
Activation of the tumor necrosis factor R1/Fas receptor results in the cleavage of cytosolic BID to truncated tBID. tBID translocates to the mitochondria to induce the oligomerization of BAX or BAK, resulting in the release of cytochrome c (Cyt c). Here we demonstrate that in tumor necrosis factor α-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex that does not include BAX or BAK. Using fluorescence resonance energy transfer analysis and co-immunoprecipitation, we demonstrate that tBID-tBID interactions occur in the mitochondria of living cells. Cross-linking experiments using a tBID-GST chimera indicated that tBID forms homotrimers in the mitochondrial membrane. To test the functional consequence of tBID oligomerization, we expressed a chimeric FKBP-tBID molecule. Enforced dimerization of FKBP-tBID by the bivalent ligand FK1012 resulted in Cyt c release, caspase activation, and apoptosis. Surprisingly, enforced dimerization of tBID did not result in the dimerization of either BAX or BAK. Moreover, a tBID BH3 mutant (G94E), which does not interact with or induce the dimerization of either BAX or BAK, formed the 45-kDa complex and induced both Cyt c release and apoptosis. Thus, tBID oligomerization may represent an alternative mechanism for inducing mitochondrial dysfunction and apoptosis.
Phosphorylation of the epithelial Na + channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the β and γ subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of β and γENaC, have been identified by an in vitro assay. One of these phosphorylates βThr-613 and γThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of γThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating βThr-613 and γThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions β613 or γ623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on βThr-613 and γThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of βThr-613 and γThr-623 down-regulates the channel by facilitating its interaction with Nedd4.
A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the β and γ subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539-13547]. One of them was identified as ERK which phosphorylates βT613 and γT623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK-2-like kinase. It phosphorylates βS631, a well-conserved serine on the β subunit. Such phosphorylation is observed both in vitro using glutathione-S-transferase-ENaC fusion proteins and in vivo in ENaC-expressing Xenopus oocytes. The γ subunit is weakly phosphorylated by this protein kinase on another residue (γT599), and the C tail of α is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel.
2001
G protein-coupled inwardly rectifying K+ channels (GIRK) play a major role in inhibitory signaling in excitable and endocrine tissues. The gating mechanism of these channels is mediated by a direct interaction of the Gβγ subunits of G protein, which are released upon inhibitory neurotransmitter receptor activation. This gating mechanism is further manifested by intracellular factors such as anionic phospholipids and Na + and Mg2+ ions. In addition to the essential role of these components for channel function, phosphorylation events can also modulate channel activity. In this study we explored the involvement of redox modulation on GIRK channel function. Extracellular application of the reducing agent dithiothreitol (DTT), but not reduced glutathione, activated GIRK channels without affecting their permeation or rectification properties. The DTT-dependent activation was found to mimic receptor activation and to act directly on the channel in a membrane delimited fashion. A critical cysteine residue located in the N-terminal cytoplasmic domain was found to be essential for DTT-dependent activation in hetero- and homotetrameric contexts. Interestingly, when mutating this cysteine residue, DTT-dependent activation was abolished, but receptor-mediated channel activation was not affected. These results suggest that intracellular redox potential can play a major role in tuning GIRK channel activity in a receptor-independent manner. This sort of redox modulation can be part of an important cellular protective mechanism against ischemic or hypoxic insults.
1. The block of the IRK1/Kir2.1 inwardly rectifying K+ channel by a Ba2+ ion is highly voltage dependent, where the ion binds approximately half-way within the membrane electrical field. The mechanism by which two distinct mutations, E125N and T141A, affect Ba2+ block of Kir2.1 was investigated using heterologous expression in Xenopus oocytes. 2. Analysis of the blocking kinetics showed that E125 and T141 affect the entry and binding of Ba2+ to the channel, respectively. Replacing the glutamate at position 125 with an asparagine greatly decreased the rate at which the Ba2+ ions enter and leave the pore. In contrast, replacing the polar threonine at position 141 with an alanine affected the entry rate of the Ba2+ ions while leaving the exit rate unchanged. 3. Acidification of the extracellular solution slowed the exit rate of the Ba2+ from the wild-type channel, but had no such effect on the Kir2.1(E125N) mutant. 4. These results thus reveal two unique roles for the amino acids at positions 125 and 141 in aiding the interaction of Ba2+ with the channel. Their possible roles in K+ permeation are discussed.
Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the γ-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on β(S620) was phosphorylated as well. The possible role of γT630 and βS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na+ and had a high basal Na+ current. Thus mutating γT630 or βS620 may limit the maximal channel activity achieved by a combination of sgk and low Na+.
G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gβγ subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gβγ. Mutations in TM2 were found that mimicked the Gβγ-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gβγ subunits but depended on PtdIns(4,5)P2. The results suggest that the TM2 region plays a key role in channel gating following Gβγ binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.
2000
Aldosterone is the major corticosteroid regulating Na+ absorption in tight epithelia and acts primarily by activating the epithelial Na+ channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+ current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514-2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgk mRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose- response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+ channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.
1999
1998
Both the α and βΓ subunits of heterotrimeric guanine nucleotide- binding proteins (G proteins) communicate signals from receptors to effectors. GβΓ subunits can regulate a diverse array of effectors, including ion channels and enzymes. Gα subunits bound to guanine diphosphate (Gα-GDP) inhibit signal transduction through GβΓ subunits, suggesting a common interface on GβΓ subunits for Gα binding and effector interaction. The molecular basis for interaction of GβΓ with effectors was characterized by mutational analysis of Gβ residues that make contact with Gα-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-β2, and β- adrenergic receptor kinase revealed the Gβ residues required for activation of each effector and provides evidence for partially overlapping domains on Gβ for regulation of these effectors. This organization of interaction regions on Gβ for different effectors and Gα explains why subunit dissociation is crucial for signal transmission through GβΓ subunits.
1997
In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe137, located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D.E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser143. We have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1 (F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein βγ subunits, wild-type or mutated G- protein α subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of Gβγ in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts; interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein- gated channels.