In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na⁺), has remained underdeveloped, even though Na⁺ is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na⁺-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (k_obs) ∼0.1 min⁻¹], and the transformation of this DNAzyme into a fluorescent sensor for Na⁺ by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na⁺ catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na⁺ over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na⁺-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na⁺ in living cells.Metal ions play crucial roles in a variety of biochemical processes. As a result, the concentrations of cellular metal ions have to be highly regulated in different parts of cells, as both deficiency and surplus of metal ions can disrupt normal functions (1–4). To better understand the functions of metal ions in biology, it is important to detect metal ions selectively in living cells; such an endeavor will not only result in better understanding of cellular processes but also novel ways to reprogram these processes to achieve novel functions for biotechnological applications.Among the metal ions in cells, sodium (Na⁺) serves particularly important functions, as changes in its concentrations influence the cellular processes of numerous living organisms and cells (5–8), such as epithelial and other excitable cells (9). As one of the most abundant metal ions in intracellular fluid (10), Na⁺ affects cellular processes by triggering the activation of many signal transduction pathways, as well as influencing the actions of hormones (11). Therefore, it is important to carefully monitor the concentrations of Na⁺ in cells. Toward this goal, instrumental analyses by atomic absorption spectroscopy (12), X-ray fluorescence microscopy (13), and ²³Na NMR (14) have been used to detect the concentration of intracellular Na⁺. However, it is difficult to use these methods to obtain real-time dynamics of Na⁺ distribution in living cells. Fluorescent sensors provide an excellent choice to overcome this difficulty, as they can provide sensitive detection with high spatial and temporal resolution. However, despite significant efforts in developing fluorescent metal ion sensors, such as those based on either genetically encoded probes or small molecular sensors, most fluorescent sensors reported so far can detect divalent metal ions such as Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ (15–21). Among the limited number of Na⁺ sensors, such as sodium-binding benzofuran isophthalate (22), Sodium Green (23), CoroNa Green/Red (24, 25), and Asante NaTRIUM Green-1/2 (26), most of them are not selective for Na⁺ over K⁺ (22–25, 27, 28) or have a low binding affinity for Na⁺ (with a K_d higher than 100 mM) (25, 27–31). Furthermore, the presence of organic solvents is frequently required to achieve the desired sensitivity and selectivity for many of the Na⁺ probes (32–34), making it difficult to study Na⁺ under physiological conditions. Therefore, it is still a major challenge to design fluorescent sensors with strong affinity for Na⁺ and high selectivity over other monovalent and multivalent metal ions that work under physiological conditions.To meet this challenge, our group and others have taken advantage of an emerging class of metalloenzymes called DNAzymes (deoxyribozymes or catalytic DNA) and turned them into metal ion probes. DNAzymes were first discovered in 1994 through a combinatorial process called in vitro selection (35). Since then, many DNAzymes have been isolated via this selection process. Among them, RNA-cleaving DNAzymes are of particular interest for metal ion sensing, due to their fast reaction rate and because the cleavage, which is catalyzed by a metal ion cofactor, can easily be converted into a detectable signal (36–38). Unlike the rational design of either small-molecule or genetically encoded protein sensors, DNAzymes with desired sensitivity and specificity for a metal ion of interest can be selected from a large library of DNA molecules, containing up to 10¹⁵ different sequences (35, 39). A major advantage of DNAzymes as metal ion sensors is that metal-selective DNAzymes can be obtained without prior knowledge of necessary metal ion binding sites or specific metal–DNA interaction (40, 41). In addition, through the in vitro selection process, metal ion binding affinity and selectivity can be improved by tuning the stringency of selection pressure and introducing negative selection against competing metal ions (39, 40). Finally, DNA is easily synthesized with a variety of useful modifications and its biocompatibility makes DNAzyme-based sensors excellent tools for live-cell imaging of metal ions. As a result, several metal-specific DNAzymes have been isolated and converted into sensors for their respective metal ion cofactors, including Pb²⁺ (35, 42, 43), Cu²⁺ (44, 45), Zn²⁺ (46), UO₂²⁺ (47), and Hg²⁺ (48). They have recently been delivered into cells for monitoring UO₂²⁺ (41, 49), Pb²⁺ (50), Zn²⁺ (51), and histidine (52) in living cells.However, in contrast to the previously reported DNAzymes with divalent metal ion selectivity, no DNAzymes have been reported to have high selectivity toward a specific monovalent metal ion. Although DNAzymes that are independent of divalent metal ions have been obtained (53–55), including those using modified nucleosides with protein-like functionalities (i.e., guanidinium and imidazole) (56–58), no DNAzyme has been found to be selective for a specific monovalent metal ion over other monovalent metal ions. For example, the DNAzyme with the highest reported selectivity for Na⁺ still binds Na⁺ over K⁺ with only 1.3-fold selectivity (54). More importantly, those DNAzymes require very high concentrations of monovalent ions (molar ranges) to function and display very slow catalytic rates (e.g., 10⁻³ min⁻¹) (53–55). The poor selectivity, sensitivity, and slow catalytic rate render these DNAzymes unsuitable for cellular detection of Na⁺, due to interference from other monovalent ions such as K⁺ (which is present in concentrations about 10-fold higher than Na⁺), and the need to image the Na⁺ rapidly.In this study, we report the in vitro selection and characterization of an RNA-cleaving DNAzyme with exceptionally high selectivity (>10,000-fold) for Na⁺ over other competing metal ions, with a dynamic range covering the physiological Na⁺ concentration range (0.135–50 mM) and a fast catalytic rate (k_obs, ∼0.1 min⁻¹). This Na⁺-specific DNAzyme was transformed into a DNAzyme-based fluorescent sensor for imaging intracellular Na⁺ in living cells, by adopting an efficient DNAzyme delivery method using a cationic polypeptide, together with a photocaging strategy to allow controllable activation of the probe inside cells.     

Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents

A simultaneous increase in cytosolic Zn²⁺ and Ca²⁺ accompanies the initiation of neuronal cell death signaling cascades. However, the molecular convergence points of cellular processes activated by these cations are poorly understood. Here, we show that Ca²⁺-dependent activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is required for a cell death-enabling process previously shown to also depend on Zn²⁺. We have reported that oxidant-induced intraneuronal Zn²⁺ liberation triggers a syntaxin-dependent incorporation of Kv2.1 voltage-gated potassium channels into the plasma membrane. This channel insertion can be detected as a marked enhancement of delayed rectifier K⁺ currents in voltage clamp measurements observed at least 3 h following a short exposure to an apoptogenic stimulus. This current increase is the process responsible for the cytoplasmic loss of K⁺ that enables protease and nuclease activation during apoptosis. In the present study, we demonstrate that an oxidative stimulus also promotes intracellular Ca²⁺ release and activation of CaMKII, which, in turn, modulates the ability of syntaxin to interact with Kv2.1. Pharmacological or molecular inhibition of CaMKII prevents the K⁺ current enhancement observed following oxidative injury and, importantly, significantly increases neuronal viability. These findings reveal a previously unrecognized cooperative convergence of Ca²⁺- and Zn²⁺-mediated injurious signaling pathways, providing a potentially unique target for therapeutic intervention in neurodegenerative conditions associated with oxidative stress.Calcium has long been recognized as a critical component of neuronal cell death pathways triggered by oxidative, ischemic, and other forms of injury (1). Indeed, Ca²⁺ deregulation has been associated with a variety of detrimental processes in neurons, including mitochondrial dysfunction (2), generation of reactive oxygen species (3), and activation of apoptotic signaling cascades (4). More recently, zinc, a metal crucial for proper cellular functioning (5), has been found to be closely linked to many of the injurious conditions in which Ca²⁺ had been thought to play a prominent role (6–10). In fact, it has been suggested that a number of deleterious properties initially attributed to Ca²⁺ may have significant Zn²⁺-mediated components (11, 12). Although it is virtually impossible to chelate, or remove, Ca²⁺ without disrupting Zn²⁺ levels (13), the introduction of techniques to monitor Ca²⁺ and Zn²⁺ simultaneously in cells (14) has made it increasingly apparent that both cations have important yet possibly distinct roles in neuronal cell death (12, 15–18). However, the relationship between the cell death signaling pathways activated by the cations is unclear, and possible molecular points of convergence between these signaling cascades have yet to be identified.Injurious oxidative and nitrosative stimuli lead to the liberation of intracellular Zn²⁺ from metal binding proteins (19). The released Zn²⁺, in turn, triggers p38 MAPK- and Src-dependent Kv2.1 channel insertion into the plasma membrane, resulting in a prominent increase in delayed rectifier K⁺ currents in dying neurons, with no change in activation voltage, ∼3 h following a brief exposure to the stimulus (20–26). The increase in Kv2.1 channels present in the membrane mediates a pronounced loss of intracellular K⁺, likely accompanied by Cl⁻ (27, 28), that facilitates apoptosome assembly and caspase activation (20, 29–34). Indeed, K⁺ efflux appears to be a requisite event for the completion of many apoptotic programs, including oxidant-induced, Zn²⁺-mediated neuronal death (21).Ca²⁺ has been suggested to regulate the p38 MAPK signaling cascade via Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)-mediated activation of the MAP3K apoptosis signaling kinase-1 (ASK-1) (35). Because ASK-1 is also required for p38-dependent manifestation of the Zn²⁺-triggered, Kv2.1-mediated enhancement of K⁺ currents (36), we hypothesized that the p38 activation cascade may provide a point of convergence between Ca²⁺ and Zn²⁺ signals following oxidative injury. Here, we report that Ca²⁺ and Zn²⁺ signals do, in fact, converge on a cellular event critical for the K⁺ current enhancement, and that CaMKII is required for this process. However, CaMKII does not act upstream of p38 activation as originally hypothesized, but instead interacts with the N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin, which we showed to be necessary for the insertion of Kv2.1-encoded K⁺ channels following an apoptotic stimulus (23).     

From the Cover: Mitochondrial calcium overload is a key determinant in heart failure

Calcium (Ca²⁺) released from the sarcoplasmic reticulum (SR) is crucial for excitation–contraction (E–C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca²⁺ is essential for optimal function of these organelles. However, Ca²⁺ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca²⁺ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca²⁺ leak causes mitochondrial Ca²⁺ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca²⁺ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca²⁺ leak, we found that leaky RyR2 channels result in mitochondrial Ca²⁺ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca²⁺ overload and dysfunction in HF.Type 2 ryanodine receptor/Ca²⁺ release channel (RyR2) and type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) are the major intracellular Ca²⁺ release channels in the heart (1–3). RyR2 is essential for cardiac excitation–contraction (E–C) coupling (2), whereas the role of IP3R2 in cardiomyocytes is less well understood (3). E–C coupling requires energy in the form of ATP produced primarily by oxidative phosphorylation in mitochondria (4–8).Both increased and reduced mitochondrial Ca²⁺ levels have been implicated in mitochondrial dysfunction and increased reactive oxygen species (ROS) production in heart failure (HF) (6, 7, 9–17). Albeit Ca²⁺ is required for activation of key enzymes (i.e., pyruvate dehydrogenase phosphatase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the tricarboxylic acid (also known as Krebs) cycle (18, 19), excessive mitochondrial Ca²⁺ uptake has been associated with cellular dysfunction (14, 20). Furthermore, the exact source of mitochondrial Ca²⁺ has not been clearly established. Given the intimate anatomical and functional association between the sarcoplasmic reticulum (SR) and mitochondria (6, 21, 22), we hypothesized that SR Ca²⁺ release via RyR2 and/or IP3R2 channels in cardiomyocytes could lead to mitochondrial Ca²⁺ accumulation and dysfunction contributing to oxidative overload and energy depletion.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca²⁺ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.Two-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca²⁺ and Na⁺ channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca²⁺/Na⁺ channel with cytosolic NH₂ and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membrane-spanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2–7) that may support such a lengthy evolutionary pedigree.In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca²⁺ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4–6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca²⁺ release from “acidic stores.” The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na⁺ channels regulated by the endolysosomal phosphoinositide PI(3,5)P₂. Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactome establishes a useful community resource as a “rosetta stone” for interrogating the cell biology of TPCs and their regulation. The dataset reveals a predomination of links between TPCs and effectors controlling membrane organization and trafficking, relevant for disease states involving lysosomal proliferation where TPC functionality may be altered (14).     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺ sensor, is unclear with respect to its cellular localization, its Ca²⁺-dependent mobilization, and its action on Ca²⁺ signaling. Confocal microscopy was used to measure Ca²⁺ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca²⁺ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca²⁺ depletion. Additionally, we found no store-operated Ca²⁺ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca²⁺ content and increased SR Ca²⁺ leak. These changes in Ca²⁺ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca²⁺ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca²⁺ leak and that these actions are independent of store-operated Ca²⁺ entry, a process that is absent in normal heart cells.Store-operated Ca²⁺ entry (SOCE) is a cellular mechanism to ensure that sufficient levels of Ca²⁺ are present in the intracellular Ca²⁺ stores to enable robust signaling (1). SOCE depends on the presence and interaction of two proteins, STIM1 (stromal interaction molecule 1) and Orai1 (a low conductance plasma/sarcolemmal Ca²⁺ channel), or their equivalents (2–5). STIM1 is an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺-sensitive protein that interacts with Orai1 to activate the channel function of Orai1, a Ca²⁺ selective channel, and thus permit Ca²⁺ entry. SOCE is clearly present in nonexcitable cells such as T lymphocytes and some excitable cells including skeletal muscle cells (4, 6–13). STIM1 is a membrane-spanning ER/SR protein with a single transmembrane domain and a luminal Ca²⁺ ([Ca²⁺]_ER/SR)-sensing domain. When luminal Ca²⁺ is low (i.e., [Ca²⁺]_ER/SR drops to less than 300 µM), then STIM1 self-aggregates and associates with Orai1 to activate it, producing a SOCE current (I_SOCE) (2, 14–16) and Ca²⁺ entry (with a reversal potential E_SOCE ∼ +50 mV or more) (17, 18). Then, as [Ca²⁺]_ER/SR increases in response to the Ca²⁺ influx, the process reverses.In adult skeletal muscle cells, Ca²⁺ influx is normally low, and it has been suggested that SOCE is needed for maintaining an appropriate level of [Ca²⁺]_ER/SR and correct Ca²⁺ signaling (6, 7, 9, 19). In skeletal muscle, it has been hypothesized that STIM1 is prelocalized in the SR terminal cisternae (6, 20) and hence can more rapidly respond to changes in [Ca²⁺]_ER/SR. The putative importance of SOCE in skeletal muscle was further supported by the observation that the skeletal muscle dysfunction is significant in STIM1-null mice where 91% (30/33) of the animals died in the perinatal period from a skeletal myopathy (6). Furthermore, in humans, STIM1 mutations were identified as a genetic cause of tubular aggregate myopathy (21).Despite the clarity of the SOCE paradigm, the canonical SOCE activation process described above does not apply to all conditions in which STIM1 and Orai1 interact. For example, in T lymphocytes, STIM1 clustering is necessary and sufficient to activate SOCE, regardless of whether [Ca²⁺]_ER/SR is low (4). When present, the STIM1 EF hand mutation causes STIM1 oligomerization and constitutive Ca²⁺ influx across the plasma membrane into cells with full Ca²⁺ stores (4). Although this is consistent with the use of STIM1 clusters and puncta to measure the activation of Orai1 (15, 16, 22, 23), it does not necessarily reflect the state of [Ca²⁺]_ER/SR. Furthermore, several small-molecule bioactive reagents, such as 2-APB and FCCP, neither of which causes [Ca²⁺]_ER/SR depletion, induce STIM1 clustering (24). Thus, STIM1 may have actions that are more complicated than simple [Ca²⁺]_ER/SR sensing and Orai1 signaling.Cardiomyocytes have been reported to have SOCE (8, 13, 25, 26) but are very different from many of the cells noted above that exhibit significant [Ca²⁺]_ER/SR depletion-sensitive Ca²⁺ entry through the Ca²⁺-selective Orai1. Cardiac ventricular myocytes are different from the other cells in that they have large, regular, and dynamic changes in [Ca²⁺]_i and robust influx and extrusion pathways across the sarcolemmal membrane. For example, it is not unusual for investigators to measure a 10–20 nA calcium current (I_Ca,L) in single cardiac ventricular myocytes that is readily extruded by the sarcolemmal Na⁺/Ca²⁺ exchanger. Because of these large fluxes, adult ventricular myocytes have no “need” for SOCE and the same logic applies to neonatal cardiomyocytes. Nevertheless, reports of SOCE in neonatal cardiac myocytes are clear (10, 12, 13). Against this background, we have attempted to determine if STIM1 is present in adult cardiomyocytes and, if so, where the protein is located, how it is mobilized, and how it may interact with other Ca²⁺ signal proteins. In the work presented here, we show that STIM1 is present but that its function in heart is distinct from the canonical SOCE behavior and does not contribute to Ca²⁺ influx through I_SOCE. Instead we show that STIM1 binds phospholamban (PLN), an endogenous SERCA2a inhibitor in the heart (27), and by doing so reduces the PLN-dependent inhibition of SERCA2a and thereby indirectly activates SERCA2a.     

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca²⁺ signals is due to a signal-enhancing analysis of the Ca²⁺ activity. The rapid stimulation-evoked Ca²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.Brain function emerges from signaling in and between neurons and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1–5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca²⁺ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7–10). Ca²⁺ responses are of interest because intracellular Ca²⁺ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca²⁺-dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca²⁺ changes (11). Astrocytic Ca²⁺ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8, 10, 12, 13). Furthermore, uncaging of Ca²⁺ in astrocytes triggers vascular responses in brain slices through specific Ca²⁺-dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca²⁺ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16, 17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca²⁺ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca²⁺ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca²⁺ responses were brief enough to be a direct consequence of synaptic excitation and correlated with stimulation-induced hemodynamic responses. Fast Ca²⁺ responses in astrocyte end-feet preceded the onset of dilatation in adjacent vessels by hundreds of milliseconds. This finding might suggest that communication at the gliovascular interface contributes considerably to neurovascular coupling.     

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons

Short-term synaptic plasticity is induced by calcium (Ca²⁺) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated Ca_V2.1 Ca²⁺ channels by Ca²⁺ sensor proteins induces facilitation of Ca²⁺ currents and synaptic facilitation in cultured neurons expressing exogenous Ca_V2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca²⁺ sensor binding site. This mutation does not alter voltage dependence or kinetics of Ca_V2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼50%. In the presence of EGTA-AM to prevent global increases in free Ca²⁺, the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca²⁺ is dependent upon regulation of Ca_V2.1 channels by Ca²⁺ sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10–20 Hz. Evidently, regulation of Ca_V2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.Modification of synaptic strength in central synapses is highly dependent upon presynaptic activity. The frequency and pattern of presynaptic action potentials regulates the postsynaptic response through diverse forms of short- and long-term plasticity that are specific to individual synapses and depend upon accumulation of intracellular Ca²⁺ (1–4). Presynaptic plasticity regulates neurotransmission by varying the amount of neurotransmitter released by each presynaptic action potential (1–5). P/Q-type Ca²⁺ currents conducted by voltage-gated Ca_V2.1 Ca²⁺ channels initiate neurotransmitter release at fast excitatory glutamatergic synapses in the brain (6–9) and regulate short-term presynaptic plasticity (3, 10). These channels exhibit Ca²⁺-dependent facilitation and inactivation that is mediated by the Ca²⁺ sensor (CaS) protein calmodulin (CaM) bound to a bipartite site in their C-terminal domain composed of an IQ-like motif (IM) and a CaM binding domain (CBD) (11–14). Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents correlate with facilitation and rapid depression of synaptic transmission at the Calyx of Held (15–18). Elimination of Ca_V2.1 channels by gene deletion prevents facilitation of synaptic transmission at the Calyx of Held (19, 20). Cultured sympathetic ganglion neurons with presynaptic expression of exogenous Ca_V2.1 channels harboring mutations in their CaS regulatory site have reduced facilitation and slowed depression of postsynaptic responses because of reduced Ca²⁺-dependent facilitation and Ca²⁺-dependent inactivation of Ca_V2.1 currents (21). The CaS proteins Ca²⁺-binding protein 1 (CaBP-1), visinin-like protein-2 (VILIP-2), and neuronal Ca²⁺ sensor-1 (NCS-1) induce different degrees of Ca²⁺-dependent facilitation and inactivation of channel activity (22–26). Expression of these different CaS proteins with Ca_V2.1 channels in cultured sympathetic ganglion neurons results in corresponding bidirectional changes in facilitation and depression of the postsynaptic response (25, 26). Therefore, binding of CaS proteins to Ca_V2.1 channels at specific synapses can change the balance of CaS-dependent facilitation and inactivation of Ca_V2.1 channels, and determine the outcome of synaptic plasticity (27). Currently, it is not known whether such molecular regulation of Ca_V2.1 by CaS proteins induces or modulates synaptic plasticity in native hippocampal synapses.To understand the functional role of regulation of Ca_V2.1 channels by CaS proteins in synaptic plasticity in vivo, we generated knock-in mice with paired alanine substitutions for the isoleucine and methionine residues in the IM motif (IM-AA) in their C-terminal domain. Here we investigated the effects of mutating this CaS regulatory site on hippocampal neurotransmission and synaptic plasticity. This mutation had no effect on basal Ca²⁺ channel function or on basal synaptic transmission. However, we found reduced short-term facilitation in response to paired stimuli in autaptic synapses in hippocampal cultures and in Schaffer collateral (SC)-CA1 synapses in acutely prepared hippocampal slices. Moreover, synaptic facilitation in mutant SC-CA1 synapses developed and decayed more slowly during trains of stimuli. These results identify a critical role for modulation of Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity, which is likely to have important consequences for encoding and transmitting information in the hippocampus.     

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na⁺-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na⁺/leucine transporter LeuT, our results suggest that Na⁺ binding at the conserved second Na⁺ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.Secondary active transporters harness the energy of ion gradients to power the uphill movement of solutes across membranes. Mitchell (1) and others (2, 3) proposed and elaborated “alternating access” mechanisms wherein the transporter transitions between two conformational states that alternately expose the substrate binding site to the two sides of the membrane. The LeuT class of ion-coupled symporters consists of functionally distinct transporters that share a conserved scaffold of two sets of five transmembrane helices related by twofold symmetry around an axis nearly parallel to the membrane (4). Ions and substrates are bound near the middle of the membrane stabilized by electrostatic interactions with unwound regions of transmembrane helix (TM) 1 and often TM6 (4). The recurrence of this fold in transporters that play critical roles in fundamental physiological processes (5, 6) has spurred intense interest in defining the principles of alternating access.Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7–11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4, 7, 9–12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outward-facing, or occluded on the basis of the accessibility of the substrate binding site (7–11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).Mhp1, an Na⁺-coupled symporter of benzyl-hydantoin (BH) from Microbacterium liquefaciens, was the first LeuT-fold member to be characterized by crystal structures purported to represent outward-facing, inward-facing, and outward-facing/occluded conformations of an alternating access cycle (8, 15). In these structures, solvent access to ligand-binding sites is defined by the relative orientation between a 4-helix bundle motif and a 4-helix scaffold motif (8). In Mhp1, alternating access between inward- and outward-facing conformations, was predicted from a computational analysis based on the inverted repeat symmetry of the LeuT fold and is referred to as the rocking-bundle model (16). The conservation of the inverted symmetry prompted proposal of the rocking-bundle mechanism as a general model for LeuT-fold transporters (16). Subsequent crystal structures of other LeuT-fold transporters (7, 9, 10) tempered this prediction because the diversity of the structural rearrangements implicit in these structures is seemingly inconsistent with a conserved conformational cycle.Another outstanding question pertains to the ion-coupling mechanism and the driving force of conformational changes. The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na⁺ ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) simulations (20, 21) and electron paramagnetic resonance (EPR) analysis (13, 22) of LeuT demonstrated that Na⁺ binding favors an outward-facing conformation although it is unclear which Na⁺ site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na⁺ in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.Here, we used site-directed spin labeling (SDSL) (24) and double electron-electron resonance (DEER) spectroscopy (25) to elucidate the conformational changes underlying alternating access in Mhp1 and define the role of ion and substrate binding in driving transition between conformations. This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13, 26–32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra- and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward- and outward-facing conformations. A major finding is that this transition is driven by substrate but not Na⁺ binding. Although the amplitudes of the observed distance changes are in overall agreement with the rocking-bundle model deduced from the crystal structures of Mhp1 (8, 15) and predicted computationally (16), we present evidence that relative movement of bundle and scaffold deviate from strict rigid body. Comparative analysis of LeuT and Mhp1 alternating access reveal how the conserved LeuT fold harnesses the energy of the Na⁺ gradient through two distinct coupling mechanisms and supports divergent conformational cycles to effect substrate binding and release.     

Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms

Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na⁺ (Na_V) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial Na_V channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.General anesthetic agents have been in use for more than 160 y. However, we still understand relatively little about their mechanisms of action, which greatly limits our ability to design safer and more effective general anesthetic agents. Ion channels of the central nervous system are known to be key targets of general anesthetic agents, as their modulation can account for the endpoints and side effects of general anesthesia (1–4). Many families of ion channels are modulated by general anesthetic agents, including ligand-gated, voltage-gated, and nongated ion channels (2, 5–7). Mammalian voltage-gated Na⁺ (Na_V) channels, which mediate the upstroke of the action potential, are regulated by numerous inhaled general anesthetic agents (8–14), which generally cause inhibition. Previous work showed that inhaled general anesthetic agents, including sevoflurane, isoflurane, desflurane, and halothane, mediate inhibition by increasing the rate of Na⁺ channel inactivation, hyperpolarizing steady-state inactivation, and slowing recovery from inactivation (11, 15–18). Inhibition of presynaptic Na_V channels in the spinal cord is proposed to lead to inhibition of neurotransmitter release, facilitating immobilization—one of the endpoints of general anesthesia (14, 19, 20). Despite the importance of Na_V channels as general anesthetic targets, little is known about interaction sites or the mechanisms of action.What is known about anesthetic sites in Na_V channels comes primarily from the local anesthetic field. Local anesthetic agent binding to Na_V channels is well characterized. These amphiphilic drugs enter the channel pore from the intracellular side, causing open-channel block (21). Investigating molecular mechanisms of mammalian Na_V channel modulation by general anesthetic agents has been complicated by the lack of high-resolution structures of these channels as a result of their large size and pseudotetrameric organization. However, the recent discovery of the smaller, tetrameric bacterial Na⁺ channel family has provided an invaluable tool to characterize the structural features of Na_V channels and investigate their interactions with general anesthetic agents at the molecular level (22, 23). Several bacterial Na⁺ channels have been crystallized (24–27). These channels have a classical domain structure in which helices S1–S4 form the voltage sensor domain (VSD), S5 and S6 form the pore, and the S4–S5 linker connects the voltage sensor to the pore domain. One notable structural feature is the presence of “fenestrations” or hydrophobic tunnels through the pore domain (24).Although crystal structures are not yet reported, the bacterial Na⁺ channel NaChBac has been extensively characterized by electrophysiology (22, 28–36). Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15, 37). These anesthetic agents reduce peak current and accelerate current decay, making it conceivable that local and general anesthetic agents could share a site of action in NaChBac. The local anesthetic binding site identified in the central cavity of the mammalian Na_V1.2 channel, which mediates open channel block, is partially conserved in NaChBac (37, 38). A recent molecular dynamics (MD) modeling study found that isoflurane, which inhibits NaChBac (15), interacts with multiple regions of this channel, including the pore, the selectivity filter, and the S4–S5 linker/S6 interface (39). Although the importance of these interactions on the modulation of mammalian Na_V channels remains to be determined, the available data indicate that NaChBac is currently one of the best starting points to investigate the mechanisms of action of sevoflurane.Here, we investigated NaChBac to gain structural insight into the mechanisms of inhaled anesthetic modulation of Na_V channels. The focus of this work is sevoflurane because this anesthetic is commonly used in clinical settings and is a known inhibitor of several mammalian Na_V channels (Na_V 1.4, 1.7, and 1.8) (11, 13). A three-pronged approach incorporating MD simulation, whole-cell patch-clamp electrophysiology, and kinetic modeling suggests that sevoflurane acts on multiple sites to alter gating and permeation. Whereas the effect on gating results from modulating activation and inactivation gating at low concentrations (0.2 mM), the permeation effect is apparent at high concentrations (2 mM) and results from open channel block (2 mM). Although the net inhibitory effect of these multisite interactions is consistent with anesthetic-induced reduction of neuronal firing, general anesthesia does not simply result from a global reduction in firing. General anesthesia depends on complex mechanisms throughout the brain, which include increases and decreases in firing (3). Thus, precisely how Na⁺ channel activation by sevoflurane fits into the global effects of anesthesia remains to be seen. The present work helps elucidate the molecular mechanism of sevoflurane action on Na_V channels.     

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca²⁺ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca²⁺-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca²⁺ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca²⁺ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca²⁺ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca²⁺, intracellular Ca²⁺ buffering, and membrane potential, by their common effect on intracellular free Ca²⁺.Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca²⁺ entry through the channel itself (6–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca²⁺, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca²⁺ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca²⁺-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca²⁺ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20–23) compared with the piezo-driven glass rod (19, 24).     

SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility

Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca²⁺ and K⁺, leading to an elevation in cytosolic Ca²⁺ critical for activation of hyperactivated swimming motility. In mice, the Ca²⁺ conductance (alkalization-activated Ca²⁺-permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K⁺ conductance (sperm pH-regulated K⁺ current, KSPER) arises from a pore-forming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca²⁺ and K⁺ conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions.Upon entry into the female reproductive tract, mammalian sperm undergo a sequence of maturational steps, collectively termed capacitation, to become competent to fertilize an egg (1, 2). Two important components of this process, thought to be shared among mammalian species, are cytosolic alkalization (3–5) and then an associated increase in cytosolic Ca²⁺ (6, 7). These events are coupled with changes in ionic fluxes in sperm membrane. Over the past 10 y, the application of patch-clamp recording to individual sperm (8) has allowed identification of ionic currents that respond to alkalization and/or Ca²⁺ (9–12). In mouse sperm, alkalization leads to activation of two sperm-specific channels, the Ca²⁺-permeable CATSPER channel (8–10, 13) and the K⁺-permeable KSPER K⁺ channel (14, 15). KSPER and CATSPER currents are also present in human sperm (16–18), although intriguingly there seem to be differences in regulation of each channel type between mice and humans (11, 12, 17).Despite the species-specific differences in the details of their regulation, KSPER and CATSPER are of central importance in sperm function and fertility in both humans and mice. In mouse sperm, KSPER and CATSPER together account for all patch-clamp measurable cation current activated by voltage and alkalization (19) and are thought to act in concert to mediate the changes in membrane cation conductance and Ca²⁺ influx that occur during the onset of capacitation (14, 20). The critical role of both KSPER and CATSPER in the mouse has been established by demonstration that genetic KO of the pore-forming subunits of each channel [KSPER (15, 21) and CATSPER (22–26)] results in infertility and the CATSPER auxiliary δ subunit is also required for fertility (27).For mouse KSPER, the pore-forming subunit is termed SLO3, encoded by the kcnu1 (slo3) gene (28). SLO3, which is exclusively expressed in testis (15, 28), is a homolog of the Ca²⁺- and voltage-activated, large-conductance (BK)-type K⁺ channel (29) and heterologously expressed SLO3 results in voltage- and alkalization-activated K⁺ currents (28). However, heterologously expressed mouse SLO3 channels differ from mouse KSPER current in important ways. Specifically, whereas pH 7 substantially activates KSPER in mouse sperm at membrane potentials (V_m) between −10 and −50 mV, activation of SLO3 channels at pH 7 is scarcely observed even at an activation potential of +100 mV (30, 31). This discrepancy raised the possibility that an additional regulatory partner of SLO3 may be present in mouse sperm. Guided by the discovery of a new type of auxiliary subunit of the BK channel (32), we recently showed that a related subunit, LRRC52 (leucine-rich-repeat-containing protein 52) is selectively expressed in sperm (33). When LRRC52 is coexpressed with SLO3, the gating range of the resulting channels at a given pH is more like that of KSPER in mouse sperm. LRRC52 protein has also been shown to be present in human sperm and, when coexpressed with human SLO3, LRRC52 results in currents with properties similar to those of human KSPER (12). To evaluate the importance of the LRRC52 subunit, we have now generated lrrc52^−/− (LRRC52 KO) mice. LRRC52 KO mice have a severe fertility deficit that is associated with a shift to more positive potentials in the KSPER current activation range in the LRRC52 KO sperm. Furthermore, measurement of the net KSPER current in sperm from two other genotypes, slo3^+/− and a slo3-eGFP, suggests that sperm reproductive capacity strongly depends on the KSPER conductance available over physiological potentials. These results confirm that LRRC52 is a critical component of the KSPER channel complex and is essential for male fertility.     

Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: Involvement of the S4/S5 linker and the S6 segment

GoSlo-SR-5-6 is a novel large-conductance Ca²⁺-activated K⁺ (BK) channel agonist that shifts the activation V_1/2 of these channels in excess of −100 mV when applied at a concentration of 10 μM. Although the structure–activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan–Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.Large-conductance Ca²⁺-activated K⁺ (BK) channels are pore-forming transmembrane proteins and are allosterically modulated by voltage and Ca²⁺ (1–5). The BKα subunits form tetramers, and both regulatory β- (6) and γ-subunits (7) can associate with the pore-forming α-subunits. Although the accessory subunits are not required for functional BK channels, they alter the sensitivity of the channels to Ca²⁺, voltage, and various channel agonists. Uniquely for K⁺ channels, each BKα subunit comprises seven transmembrane (S0–S6) domains, which contain voltage-sensing residues in S1–S4 (8–10) and the pore gate domain is located in S5–S6 (11). The transmembrane domain is attached to a large intracellular domain, which comprises two regulators of conductance for K⁺ (RCK) domains (12). The RCK1 domain contains a high-affinity Ca²⁺-binding site and a low-affinity cation-binding site, which senses Mg²⁺ and high concentrations of Ca²⁺ (13, 14). Another high-affinity Ca²⁺-binding site, called the Ca²⁺ bowl (13–16), is found in the RCK2 domain. Ca²⁺ binding through these domains is transduced to the transmembrane domain via the S6/RCK1 linker (12, 17). Recent evidence (18) supports the idea that the cytosolic domain of this channel is responsible for sensing Ca²⁺, because truncated BK channels lacking the C terminus are insensitive to Ca²⁺.BK channels play a number of important roles that govern the excitability of neuronal and smooth muscle cells. In bladder smooth muscle, for example, they contribute significantly to the repolarization phase of the action potential and thus modulate the contractile activity of this tissue (19). Interestingly, BKα knockout mice (20) display a functionally incontinent phenotype, presumably due to detrusor overactivity. Furthermore, a number of studies (21, 22) have suggested that the expression of BK channels is reduced in patients suffering from neurogenic detrusor overactivity. These results suggest that BK channel activators could represent a novel therapeutic approach for treating overactive bladder. However, despite the development of a large number of BK channel openers over the last two decades (23–29), they have failed to progress through clinical trials, because they showed poor efficacy, presumably due to their lack of effect at physiological membrane potentials, combined with a reduction in BK channels in patients with overactive bladder (30).Recently, we synthesized a novel group of BK channel openers called the GoSlo-SR family (31). GoSlo-SR-5-6 (GoSlo) (10 μM) shifted the voltage dependence of activation in excess of −100 mV. Although the structure–activity relationships of these compounds has been established (31, 32), little is known about their mode of action on BK channels, other than GoSlo does not require the β₁-subunit to mediate its effects (33).The purpose of the present study was to examine the molecular mechanisms underlying the excitatory effects of GoSlo on BK channels expressed in HEK cells. Recently, a number of studies have demonstrated that BK channel openers such as Cym04, NS1619, and omega-3 fatty acids mediate their effects, at least partially, through an interaction with the S6/RCK1 linker (34) or the S6 segment (35) of the BK channel. Our results demonstrate that GoSlo mediates its effects by interactions with S6 and the S4/S5 linker (S4S5_L), and this was reduced by combined 317, 326, and 227 mutations. The Horrigan–Aldrich (HA) allosteric model of BK channel gating (5) suggests that GoSlo enhanced the equilibrium constants for both pore opening and voltage sensor activation but reduced the voltage sensor/gate coupling.     

Nonfunctional NaV1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura. Mutations causing FHM type 3 have been identified in SCN1A, the gene encoding the Na_v1.1 Na⁺ channel, which is also a major target of epileptogenic mutations and is particularly important for the excitability of GABAergic neurons. However, functional studies of Na_V1.1 FHM mutations have generated controversial results. In particular, it has been shown that the Na_V1.1-L1649Q mutant is nonfunctional when expressed in a human cell line because of impaired plasma membrane expression, similarly to Na_V1.1 mutants that cause severe epilepsy, but we have observed gain-of-function effects for other Na_V1.1 FHM mutants. Here we show that Na_V1.1-L1649Q is nonfunctional because of folding defects that are rescuable by incubation at lower temperatures or coexpression of interacting proteins, and that a partial rescue is sufficient for inducing an overall gain of function because of the modifications in gating properties. Strikingly, when expressed in neurons, the mutant was partially rescued and was a constitutive gain of function. A computational model showed that 35% rescue can be sufficient for inducing gain of function. Interestingly, previously described folding-defective epileptogenic Na_V1.1 mutants show loss of function also when rescued. Our results are consistent with gain of function as the functional effect of Na_V1.1 FHM mutations and hyperexcitability of GABAergic neurons as the pathomechanism of FHM type 3.Epilepsy and migraine are common neurologic disorders that may have pathophysiological links (1–3). Mutations have been identified for some rare types of epilepsy and migraine (1, 4–6), opening a window for investigating their pathogenic mechanisms, which may provide useful information also about more common forms. The Na⁺ channel α subunit Na_V1.1, encoded by the SCN1A gene, is the target of hundreds of epileptogenic mutations (7–9), and of mutations causing familial hemiplegic migraine type 3 (FHM-3), a rare subtype of migraine with aura characterized by hemiplegia during the attacks, which can also be caused by mutations of Ca_V2.1 Ca²⁺ channels and the α2 subunit of the Na⁺/K⁺ ATPase (FHM types 1 and 2) (1, 6). The results of most studies suggest that epileptogenic Na_V1.1 mutations cause variable degrees of loss of function of Na_V1.1, leading to reduced Na⁺ current and excitability in GABAergic neurons, and resulting in decreased inhibition in neuronal networks (10–14). The most severe phenotypes (e.g., Dravet syndrome, an extremely severe epileptic encephalopathy) are in general caused by mutations that induce complete Na_V1.1 loss of function, leading to haploinsufficiency (15). Thus, it has been hypothesized that a more severe loss of function would cause more severe epilepsy (8). Functional studies of Na_V1.1 FHM mutations have generated more confusing results (1). For instance, we have reported gain-of-function effects for the mutant Q1489K causing pure FHM (16), and modulable gain-/loss-of-function effects for the mutant T1174S associated with FHM or mild epilepsy in different branches of the family (17). Overall, our results are consistent with a gain of function of Na_V1.1 as the cause of FHM, which might induce cortical spreading depression (CSD), a probable pathomechanism of migraine, because of hyperexcitability of GABAergic interneurons (16). However, a study has reported loss of function for FHM hNa_V1.1 mutants expressed in the human cell line tsA-201—in particular, complete loss of function for the L1649Q mutant because of lack of cell surface expression (18). L1649Q has been identified in a four-generation family with eight members presenting with FHM, without epilepsy or other neurologic symptoms (19); this is a puzzling result more consistent with a phenotype of severe epilepsy (7, 8). We have found that Na_V1.1 epileptogenic mutations can induce loss of function by causing folding defects (20), which can be partially rescued by incubation of the transfected cells at lower temperatures (≤30 °C) or by molecular interactions (21, 22), as recently confirmed also for other epileptogenic Na_V1.1 mutants (23, 24). We report here that L1649Q is a folding-defective mutant that, when partially rescued, is characterized by an overall gain of function, consistent with our hypothesis of FHM type 3 pathomechanism (16).     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl? channels

Ca²⁺-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca²⁺ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP)—we discovered that Ca²⁺-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca²⁺-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM₁₂₃₄. These effects are Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca²⁺-activated chloride channels.Calcium (Ca²⁺)-activated chloride (Cl^–) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3–5), smooth muscle contraction (6), olfactory transduction (7, 8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).Three laboratories independently identified the transmembrane protein TMEM16A as the molecular component of a CaCC (12–14). TMEM16A belongs to a protein family with 10 members encoded by distinct genes (15–18). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2, 12–14, 19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20–22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2, 19). Human TMEM16A has four alternatively spliced segments (a–d), differential inclusion of which modify voltage and Ca²⁺ sensitivity of resultant channel splice variants (24).CaCCs are highly sensitive to intracellular [Ca²⁺], displaying graded increases in Cl⁻ current (I_Cl) amplitude as [Ca²⁺]_i is raised from resting levels (∼100 nM) to the 1- to 2-μM range. In some cases, high [Ca²⁺]_i (>10 μM) leads to decreased I_Cl amplitude (inactivation) (25–27). The Ca²⁺ sensor(s) for Ca²⁺-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is unknown. There are two possible nonexclusive mechanisms: (i) direct Ca²⁺ binding to the channel or (ii) Ca²⁺ binding through a separate Ca²⁺-sensing protein. The TMEM16A sequence does not reveal any canonical Ca²⁺-binding EF hand motifs (14, 16, 17). A sequence in the first intracellular loop of TMEM16A resembling the “Ca²⁺ bowl” in large conductance Ca²⁺-activated K⁺ (BK) channels was disqualified by mutagenesis as the Ca²⁺ sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca²⁺ sensitivity of TMEM16A (29, 30).Some reports have suggested involvement of calmodulin (CaM) in distinct aspects of Ca²⁺-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segment b (31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is contradicted by the robust CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segment b (2, 24). Recently, Ca²⁺–CaM was found to bind TMEM16A(ac) in a Ca²⁺-dependent manner and result in an increased permeability of the channel to HCO₃⁻ (32). Deleting the Ca²⁺–CaM binding site did not affect CDA of TMEM16A(ac). Ca²⁺–CaM regulation of TMEM16A HCO₃⁻ permeability conforms to a traditional signaling mode where Ca²⁺ binds to freely diffusing CaM to form a Ca²⁺–CaM complex that then interacts with a target protein.There are several examples of an alternative mode of CaM signaling in which Ca²⁺-free CaM (apoCaM) is preassociated with target proteins under resting [Ca²⁺]_i conditions and acts as a resident Ca²⁺ sensor to regulate function of the host protein in response to increased [Ca²⁺]_i (33). This mode of CaM signaling is used as the activating mechanism for small conductance K⁺ channels (34) and Ca²⁺-dependent regulation of high voltage-gated Ca²⁺ (Ca_V1 and Ca_V2) channels (35, 36). A distinguishing feature of this mode of CaM signaling is that it is impervious to pharmacological inhibitors of Ca²⁺–CaM, but can be eliminated in dominant negative fashion by a CaM mutant, CaM₁₂₃₄, in which all four EF hands have been mutated so they no longer bind Ca²⁺ (34, 36). Whether apocalmodulin preassociates with TMEM16A/TMEM16B channel complexes and participates in Ca²⁺-dependent regulation of these channels is controversial (30, 37, 38).Using a recently developed bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP) (39)—we discovered that apoCaM is functionally preassociated with TMEM16A/16B channel complexes. Whereas the resident apoCaM is not necessary for CDA, it does mediate two distinct Ca²⁺-dependent processes in TMEM16A. First, it causes a leftward shift in the Ca²⁺-dependent activation curve at [Ca²⁺]_i ≤ 1 μM, an effect we term Ca²⁺-dependent “sensitization” of activation (CDSA). Second, it is the Ca²⁺ sensor for CDI observed at [Ca²⁺]_i > 10 μM. The two opposite effects are independently mediated by the two lobes of preassociated apoCaM— Ca²⁺ occupancy of the N lobe leads to CDI, whereas the C lobe mediates CDSA. Alternative splicing of TMEM16A provides a mechanism for regulating apoCaM binding and signaling. TMEM16A splice variants lacking segment a lost apoCaM binding altogether, eliminating both CDSA and CDI, whereas variants specifically lacking segment b were selectively deficient in CDI. Finally, TMEM16A variants defective in apoCaM binding displayed dramatically decreased trafficking to the cell surface.