Localization of the tubby domain, a PI(4,5)P2 biosensor, to E-Syt3-rich endoplasmic reticulum-plasma membrane junctions.

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER-PM junctions) as shown by colocalization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs.

A versatile functional interaction between electrically silent KV subunits and KV7 potassium channels.

Voltage-gated K+ (KV) channels govern K+ ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our findings demonstrate that KV cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types.

SLC26 Anion Transporters.

Solute carrier family 26 (SLC26) is a family of functionally diverse anion transporters found in all kingdoms of life. Anions transported by SLC26 proteins include chloride, bicarbonate, and sulfate, but also small organic dicarboxylates such as fumarate and oxalate. The human genome encodes ten functional homologs, several of which are causally associated with severe human diseases, highlighting their physiological importance. Here, we review novel insights into the structure and function of SLC26 proteins and summarize the physiological relevance of human members.

ß-Secretase BACE1 Is Required for Normal Cochlear Function.

Cleavage of amyloid precursor protein (APP) by ß-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-ß peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1-/- mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1-/- mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1-/- mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1-/- mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENT Given its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Identification of Cav2-PKCß and Cav2-NOS1 complexes as entities for ultrafast electrochemical coupling.

Voltage-activated calcium (Cav) channels couple intracellular signaling pathways to membrane potential by providing Ca2+ ions as second messengers at sufficiently high concentrations to modulate effector proteins located in the intimate vicinity of those channels. Here we show that protein kinase Cß (PKCß) and brain nitric oxide synthase (NOS1), both identified by proteomic analysis as constituents of the protein nano-environment of Cav2 channels in the brain, directly coassemble with Cav2.2 channels upon heterologous coexpression. Within Cav2.2-PKCß and Cav2.2-NOS1 complexes voltage-triggered Ca2+ influx through the Cav channels reliably initiates enzymatic activity within milliseconds. Using BKCa channels as target sensors for nitric oxide and protein phosphorylation together with high concentrations of Ca2+ buffers showed that the complex-mediated Ca2+ signaling occurs in local signaling domains at the plasma membrane. Our results establish Cav2-enzyme complexes as molecular entities for fast electrochemical coupling that reliably convert brief membrane depolarization into precisely timed intracellular signaling events in the mammalian brain.

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing.

Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.

A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN.

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN's substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.

Discovery and functional characterization of a neomorphic PTEN mutation.

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.

Anion translocation through an Slc26 transporter mediates lumen expansion during tubulogenesis.

Lumen formation is a critical event in biological tube formation, yet its molecular mechanisms remain poorly understood. Specifically, how lumen expansion is coordinated with other processes of tubulogenesis is not well known, and the role of membrane transporters in tubulogenesis during development has not been adequately addressed. Here we identify a solute carrier 26 (Slc26) family protein as an essential regulator of tubulogenesis using the notochord of the invertebrate chordate Ciona intestinalis as a model. Ci-Slc26aα is indispensable for lumen formation and expansion, but not for apical/luminal membrane formation and lumen connection. Ci-Slc26aα acts as an anion transporter, mediating the electrogenic exchange of sulfate or oxalate for chloride or bicarbonate and electroneutral chloride:bicarbonate exchange. Mutant rescue assays show that this transport activity is essential for Ci-Slc26aα's in vivo function. Our work reveals the consequences and relationships of several key processes in lumen formation, and establishes an in vivo assay for studying the molecular basis of the transport properties of SLC26 family transporters and their related diseases.

Differential phospholipase C-dependent modulation of TASK and TREK two-pore domain K+ channels in rat thalamocortical relay neurons.

KEY POINTS: During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K(+) channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus. The activity of two-pore domain potassium channels (K2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K(+) channel (TWIK)-related acid-sensitive K(+) (TASK) channels and TWIK-related K(+) (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCß), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2 ) and diacylglycerol (DAG) acting downstream of PLCß was probed. The standing outward current (ISO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCß as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MAChR stimulation.

A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity.

Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.

Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing.

In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, we generated and analyzed dpres mutant flies. We found that dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus we conclude that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, we show that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification.

Chloride binding and cholesterol effects on high frequency complex nonlinear capacitance (cNLC) in the mouse outer hair cell: experiment and molecular dynamics.

The function of prestin (SLC26a5), an anion transport family member, has evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. The frequency extent of this boost is governed by the voltage-dependent kinetics of the protein's charge movements, otherwise known as nonlinear capacitance (NLC) that we measure in membrane patches under voltage clamp. Here we extend our previous studies on guinea pig OHCs by studying the frequency response of NLC in the mouse OHC, a species with higher frequency auditory needs. We find that the characteristic frequency cut-off (F is ) for the mouse surpasses that of the guinea pig, being 27 kHz vs. 19 kHz, respectively; nevertheless, each shows significant activity in the ultrasonic range. We also evaluate the influence of anion binding on prestin frequency response. Several single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack anion influence. In agreement, we show absence of anion binding through molecular dynamics (MD) simulations. NLC F is in the S396E knock-in mouse remains the same as controls, indicating that high frequency activity is likely governed by viscoelastic loads within the membrane characterized by stretched-exponential frequency roll-off. Accordingly, treatment with MßCD, which removes membrane cholesterol, possibly from prestin itself, and can alter membrane fluidity, augments NLC F is out to 39 kHz. Although interactions between membrane lipid and prestin have been suggested from structural studies to arise at their interfacial boundaries within the membrane, our MD simulations suggest that phospholipids can insert within transmembrane domains of prestin during voltage perturbation. Such novel lipid-protein interactions could account for our observed changes in the phase of prestin's voltage-sensor charge movements across frequency. We hypothesize that because prestin tertiary structures of all species studied to-date are indistinguishable, it is likely that any special auditory requirements of individual species for cochlear amplification have evolved to capitalize on prestin performance by modifying, not the protein itself, but the external loads on the protein, including those within the membrane and organ of Corti. Significance: Prestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities, yet its tertiary structure is indistinguishable among those species studied. We find that prestin kinetics is faster in mice than in guinea pigs, mice showing higher frequency auditory capabilities. Chloride binding is not influential, but membrane lipids/viscosity is. We suggest that the evolution of prestin's species performance involves modifications of impinging loads, not the protein itself.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.

Distribution of ciliary adaptor proteins tubby and TULP3 in the organ of Corti.

Tubby-like proteins are membrane-associated adaptors that mediate directional trafficking into primary cilia. In inner ear sensory epithelia, cilia-including the hair cell's kinocilium-play important roles as organizers of polarity, tissue architecture and cellular function. However, auditory dysfunction in tubby mutant mice was recently found to be related to a non-ciliary function of tubby, the organization of a protein complex in sensory hair bundles of auditory outer hair cells (OHCs). Targeting of signaling components into cilia in the cochlea might therefore rather rely on closely related tubby-like proteins (TULPs). In this study, we compared cellular and subcellular localization of tubby and TULP3 in the mouse inner ear sensory organs. Immunofluorescence microscopy confirmed the previously reported highly selective localization of tubby in the stereocilia tips of OHCs and revealed a previously unnoticed transient localization to kinocilia during early postnatal development. TULP3 was detected in the organ of Corti and vestibular sensory epithelium, where it displayed a complex spatiotemporal pattern. TULP3 localized to kinocilia of cochlear and vestibular hair cells in early postnatal development but disappeared subsequently before the onset of hearing. This pattern suggested a role in targeting ciliary components into kinocilia, possibly related to the developmental processes that shape the sensory epithelia. Concurrent with loss from kinocilia, pronounced TULP3 immunolabeling progressively appeared at microtubule bundles in non-sensory Pillar (PCs) and Deiters cells (DC). This subcellular localization may indicate a novel function of TULP proteins associated with the formation or regulation of microtubule-based cellular structures.

Elevator-like movements of prestin mediate outer hair cell electromotility.

The outstanding acuity of the mammalian ear relies on cochlear amplification, an active mechanism based on the electromotility (eM) of outer hair cells. eM is a piezoelectric mechanism generated by little-understood, voltage-induced conformational changes of the anion transporter homolog prestin (SLC26A5). We used a combination of molecular dynamics (MD) simulations and biophysical approaches to identify the structural dynamics of prestin that mediate eM. MD simulations showed that prestin samples a vast conformational landscape with expanded (ES) and compact (CS) states beyond previously reported prestin structures. Transition from CS to ES is dominated by the translational-rotational movement of prestin's transport domain, akin to elevator-type substrate translocation by related solute carriers. Reversible transition between CS and ES states was supported experimentally by cysteine accessibility scanning, cysteine cross-linking between transport and scaffold domains, and voltage-clamp fluorometry (VCF). Our data demonstrate that prestin's piezoelectric dynamics recapitulate essential steps of a structurally conserved ion transport cycle.

A human phospholipid phosphatase activated by a transmembrane control module.

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.

Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

Progress in understanding the structural mechanism underlying prestin's electromotile activity.

Prestin (SLC26A5), a member of the SLC26 transporter family, is the molecular actuator that drives OHC electromotility (eM). A wealth of biophysical data indicates that eM is mediated by an area motor mechanism, in which prestin molecules act as elementary actuators by changing their area in the membrane in response to changes in membrane potential. The area changes of a large and densely packed population of prestin molecules sum up, resulting in macroscopic cellular movement. At the single protein level, this model implies major voltage-driven conformational rearrangements. However, the nature of these structural dynamics remained unknown. A main obstacle in elucidating the eM mechanism has been the lack of structural information about SLC26 transporters. The recent emergence of several high-resolution cryo-EM structures of prestin as well as other SLC26 transporter family members now provides a reliable picture of prestin's molecular architecture. Thus, SLC26 transporters including prestin generally are dimers, and each protomer is folded according to a 7+7 transmembrane domain inverted repeat (7TMIR) architecture. Here, we review these structural findings and discuss insights into a potential molecular mechanism. Most important, distinct conformations were observed when purifying and imaging prestin bound to either its physiological ligand, chloride, or to competitively inhibitory anions, sulfate or salicylate. Despite differences in detail, these structural snapshots indicate that the conformational landscape of prestin includes rearrangements between the two major domains of prestin's transmembrane region (TMD), core and scaffold ('gate') domains. Notably, distinct conformations differ in the area the TMD occupies in the membrane and in their impact on the immediate lipid environment. Both effects can contribute to generate membrane deformation and thus may underly electromotility. Further functional studies will be necessary to determine whether these or similar structural rearrangements are driven by membrane potential to mediate piezoelectric activity. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.