Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) Ca²⁺ transporters pump cytosolic Ca²⁺ into the endoplasmic reticulum, maintaining a Ca²⁺ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca²⁺ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca²⁺ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca²⁺ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca²⁺-bound E1 conformation and alters Ca²⁺-transport kinetics, which provides the rationale for the higher apparent Ca²⁺ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na⁺,K⁺-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca²⁺ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.     

CaBP1, a neuronal Ca2+ sensor protein,inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca²⁺ channels, including inositol 1,4,5-trisphosphate receptors (InsP₃Rs). CaBP1 alone does not affect InsP₃R activity, but it inhibits InsP₃-evoked Ca²⁺ release by slowing the rate of InsP₃R opening. The inhibition is enhanced by Ca²⁺ binding to both the InsP₃R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP₃R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca²⁺ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP₃-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP₃R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP₃R pore. InsP₃ activates InsP₃Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP₃-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP₃ attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP₃R activity by restricting the intersubunit movements that initiate gating.     

Presenilin 2 modulates endoplasmic reticulum (ER)-mitochondria interactions and Ca2+ cross-talk

Presenilin mutations are the main cause of familial Alzheimer's disease (FAD). Presenilins also play a key role in Ca(2+) homeostasis, and their FAD-linked mutants affect cellular Ca(2+) handling in several ways. We previously have demonstrated that FAD-linked presenilin 2 (PS2) mutants decrease the Ca(2+) content of the endoplasmic reticulum (ER) by inhibiting sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity and increasing ER Ca(2+) leak. Here we focus on the effect of presenilins on mitochondrial Ca(2+) dynamics. By using genetically encoded Ca(2+) indicators specifically targeted to mitochondria (aequorin- and GFP-based probes) in SH-SY5Y cells and primary neuronal cultures, we show that overexpression or down-regulation of PS2, but not of presenilin 1 (PS1), modulates the Ca(2+) shuttling between ER and mitochondria, with its FAD mutants strongly favoring Ca(2+) transfer between the two organelles. This effect is not caused by a direct PS2 action on mitochondrial Ca(2+)-uptake machinery but rather by an increased physical interaction between ER and mitochondria that augments the frequency of Ca(2+) hot spots generated at the cytoplasmic surface of the outer mitochondrial membrane upon stimulation. This PS2 function adds further complexity to the multifaceted nature of presenilins and to their physiological role within the cell. We also discuss the importance of this additional effect of FAD-linked PS2 mutants for the understanding of FAD pathogenesis.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.     

Regulation of sarcolemmal Ca2+ pump by endogenous protein phosphatases

Summary The function of several key sarcolemmal proteins is modulated through phosphorylation-dephosphorylation of serine/threonine residues. While the involvement of sarcolemma-associated protein kinases in the phosphorylation of these proteins has been established, the nature of the protein phosphatases controlling these proteins has not been investigated. Rat heart sarcolemma contains two protein phosphatase isozymes, protein phosphatase 1 and 2A, which are distinguished on the basis of their susceptibility of inhibitor 2. Both isozymes elute from a Bio Gel A-0.5 column in association with the highest molecular weight protein fraction. However, some protein phosphatase 1 activity elutes with a smaller molecular weight fraction of about 37000, suggesting that the native enzyme exists as a catalytic subunit in complex with an anchor protein. Inhibition of the protein phosphatases using standard inhibitors leads to a stimulation in both the rate and extent of³²P incorporation into isolated sarcolemma. Also affected by inhibition of protein phosphatase activity is the rate of ATP-dependent calcium uptake, which is stimulated following exposure to either inhibitor 2, a classical protein phosphatase 1 inhibitor, and microcystin, a protein phosphatase 1 and 2A inhibitor. The data suggest that the protein phosphatases regulate the dephosphorylation of sarcolemmal proteins Through this mechanism they serve as important modulators of the sarcolemmal Ca²⁺ pump.     

Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels

The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Relationship between Ca2+-affinity and shielding of bulk water in the Ca2+-pump from molecular dynamics simulations

The sarcoplasmic reticulum Ca(2+)-ATPase transports two Ca(2+) per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for Ca(2+) by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type Ca(2+)-ATPase in the Ca(2+)-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca(2+), but only Glu771Gln completely looses the Ca(2+)-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca(2+) was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca(2+) and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of Ca(2+) in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca(2+) and point to a strong relationship between Ca(2+)-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.     

Ion sensing in the RCK1 domain of BK channels

BK-type K(+) channels are activated by voltage and intracellular Ca(2+), which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca(2+) binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca(2+) binding site. This site is specific for binding to Ca(2+) but not Cd(2+). Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca(2+) by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca(2+) binding at this site opens the activation gate of the channel that is remotely located in the membrane.     

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections

Calcium (Ca(2+)) release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca(2+) events ("sparks") that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP(3)Rs. Here, we have identified spatially fixed, local Ca(2+) release events ("pulsars") in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca(2+) pulsars are mediated by IP(3)Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP(3) by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca(2+) pulsars, whereas blunting IP(3) production, blocking IP(3)Rs, or depleting endoplasmic reticulum Ca(2+) inhibited these events. The elementary properties of Ca(2+) pulsars were distinct from ryanodine-receptor-mediated Ca(2+) sparks in smooth muscle and from IP(3)-mediated Ca(2+) puffs in Xenopus oocytes. The intermediate conductance, Ca(2+)-sensitive potassium (K(Ca)3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca(2+) pulsars also depolarized to a similar extent, and blocking K(Ca)3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP(3) signaling in which Ca(2+) release is spatially restricted to transmit intercellular signals.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini

BACKGROUND & AIMS: The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are the principal Ca2+-release channels in cells and are believed to serve distinct roles in cytosolic Ca2+ (Ca(i)2+) signaling. This study investigated whether these receptors instead can release Ca2+ in a coordinated fashion. METHODS: Apical and basolateral Ca(i)2+ signals were monitored in rat pancreatic acinar cells by time-lapse confocal microscopy. Caged forms of second messengers were microinjected into individual cells and then photoreleased in a controlled fashion by either UV or 2-photon flash photolysis. RESULTS: InsP3 increased Ca(i)2+ primarily in the apical region of pancreatic acinar cells, whereas the RyR agonist cyclic adenosine diphosphate ribose (cADPR) increased Ca(i)2+ primarily in the basolateral region. Apical-to-basal Ca(i)2+ waves were induced by acetylcholine and initiation of these waves was blocked by the InsP3R inhibitor heparin, whereas propagation into the basolateral region was inhibited by the cADPR inhibitor 8-amino-cADPR. To examine integration of apical and basolateral Ca(i)2+ signals, Ca2+ was selectively released either apically or basolaterally using 2-photon flash photolysis. Ca(i)2+ increases were transient and localized in unstimulated cells. More complex Ca(i)2+ signaling patterns, including polarized Ca(i)2+ waves, were observed when Ca2+ was photoreleased in cells stimulated with subthreshold concentrations of acetylcholine. CONCLUSIONS: Polarized Ca(i)2+ waves are induced in acinar cells by serial activation of apical InsP3Rs and then basolateral RyRs, and subcellular release of Ca2+ coordinates the actions of these 2 types of Ca2+ channels. This subcellular integration of Ca2+-release channels shows a new level of complexity in the formation of Ca(i)2+ waves.     

Reduced expression of sarcalumenin and related Ca2+ -regulatory proteins in aged rat skeletal muscle

In skeletal muscle, Ca(2+)-cycling through the sarcoplasm regulates the excitation-contraction-relaxation cycle. Since uncoupling between sarcolemmal excitation and fibre contraction may play a key role in the functional decline of aged muscle, this study has evaluated the expression levels of key Ca(2+)-handling proteins in senescent preparations using immunoblotting and confocal microscopy. Sarcalumenin, a major luminal Ca(2+)-binding protein that mediates ion shuttling in the longitudinal sarcoplasmic reticulum, was found to be greatly reduced in aged rat tibialis anterior, gastrocnemius and soleus muscle as compared to adult specimens. Minor sarcolemmal components of Ca(2+)-extrusion, such as the surface Ca(2+)-ATPase and the Na(+)-Ca(2+)-exchanger, were also diminished in senescent fibres. No major changes were observed for calsequestrin, sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor Ca(2+)-release channel. In contrast, the age-dependent reduction in the alpha(1S)-subunit of the dihydropryridine receptor was confirmed. Hence, this report has shown that downstream from the well-established defect in coupling between the t-tubular voltage sensor and the junctional Ca(2+)-release channel complex, additional age-related alterations exist in the expression of essential Ca(2+)-handling proteins. This may trigger abnormal luminal Ca(2+)-buffering and/or decreased plasmalemmal Ca(2+)-removal, which could exacerbate impaired signaling or disturbed intracellular ion balance in aged fibres, thereby causing contractile weakness.     

Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells

Angiostensin II (Ang II) regulates the migration and proliferation of vascular smooth muscle cells. Recent studies indicate that intermediate-conductance Ca2+ -activated K+ (IKca) channels have an important role in cell migration and proliferation. It is not known, however, whether the action of Ang II is linked to IKca channel regulation. Here, we investigated the modulation of IKca channels by Ang II in artery smooth muscle cells. Functional IKca channel expression in cultured embryonic rat aorta smooth muscle (A10) cells was studied using the patch-clamp technique. These cells predominantly express IKca channels. In contrast, large-conductance Ca2+ -activated K+ (BKca) currents were rarely observed in excised patches. Ang II increased the IKca current in a contration-dependent manner. Losartan (1.0 microM), an AT1 selective antagonist, abolished the activation of IKca channels by Ang II. Pretreatment with 100 microM myristoylated protein kinase C inhibitor peptide 20-28 or 10 microM GF109203X completely abolished the AngII-induced activation of IKca currents, whereas the action of Ang II was not prevented in the presence of 100 microM Rp-cyclic 3', 5'-hydrogen phosphotiate adenosine triethylammonium, a protein kinase A inhibitor, or 1.0 microM KT-5823, a protein kinase G inhibitor. A membrane permeant analogue of diacylglycerol 1, 2-dioctanoyl-sn-glycerol (10 microM) induced the activation of IKca currents. These data suggest that Ang II activates IKca channels through the activation of protein kinase C, and the AT1 receptor is involved in the regulation of these channels.     

Allosteric modulation of alternatively spliced Ca2+-activated Cl− channels TMEM16A by PI(4,5)P2 and CaMKII

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl⁻ channel activated by intracellular Ca²⁺ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P₂ degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P₂ depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P₂ sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (P_O). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P₂, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P₂. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P₂-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P₂, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

TMEM16A (anoctamin1) plays a wide range of physiological roles in diverse cell types, including contraction of smooth muscle and gastrointestinal motility, secretion of Cl⁻ in epithelial cells, detection of noxious heat in nociceptive neurons, modulation of neuronal excitability, and regulation of cell volume (1). TMEM16A channels, from a family of 10 anoctamin proteins (TMEM16A–K), continuously monitor the concentration of intracellular Ca²⁺ and function as Ca²⁺-activated Cl⁻ channels (2–4). Several splice variants of TMEM16A generated by combinatorial inclusion or exclusion of four exon segments, a, b, c, and d (5–7), display unique electrophysiological properties in tissues. Segments a and b lie in the N terminus, and segments c and d lie in the first intracellular loop of TMEM16A. Among the four segments, it is known that b and c help regulate the cytosolic Ca²⁺ sensitivity and voltage dependence of channel gating. For example, inclusion of the b-segment results in decreased channel sensitivity to intracellular Ca²⁺ rise, whereas skipping of the c-segment reduces channel activity and also impairs Ca²⁺ sensitivity (5, 8, 9). In addition to inclusion or skipping of each segment, calmodulin (10–13), phosphorylation (14–16), protons (17–19), and lipids (20–27) also impact on the gating of TMEM16A channels.Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a key signaling phospholipid in the inner leaflet of the plasma membrane. It acts as a cofactor that regulates many types of ion channels and receptors (28–30), and thus depletion of membrane PI(4,5)P₂ by the activation of either phospholipase C (PLC) or phosphoinositide 5-phosphatases leads to decreases or increases in gating activity of ion channels. Of the TMEM16 family, TMEM16A, TMEM16B, and TMEM16F are ion channels best known to be modulated by PI(4,5)P₂ (21–27, 31). Several studies showed that PI(4,5)P₂ is required for sustained TMEM16A channel activity and stabilizes the Ca²⁺-bound open state of the channels (23, 24, 32). Further work located a PI(4,5)P₂ regulatory region and demonstrated how PI(4,5)P₂ interacts with TMEM16A to regulate channel gating by performing computational simulation. Le et al. (25) proposed that channel activation and desensitization are mediated by two distinct structural modules; one is a PI(4,5)P₂-binding module formed by putative PI(4,5)P₂-binding residues of TMs 3–5 located near the cytoplasmic membrane interface and another is a Ca²⁺-binding module of TMs 6–8 involved in the primary opening of the channel pore by Ca²⁺. Yu et al. (26) identified three key binding sites involved in TMEM16A–PI(4,5)P₂ interaction. When PI(4,5)P₂ interacts with these binding residues, which form networks with each other, it affects TMEM16A channel gating as a result of the conformational change of TM6.In our study, using exogenous lipid phosphatase tools and mutagenesis, we found that PI(4,5)P₂ differentially regulates channel activity depending on the TMEM16A splice variant. In addition, we found that the presence or absence of intracellular ATP is a key determinant of the PI(4,5)P₂ sensitivity of TMEM16A. Through structural analysis partly based on a recent cryogenic electron microscopy (cryo-EM) structure of TMEM16A, we also confirmed that phosphorylation of serine 673 by CaMKII allosterically regulates the structure of a PI(4,5)P₂ interaction site in the RDR domain of TMEM16A(ac) near to transmembrane segment 3 (TM3). Together, our data reveal a molecular mechanism of TMEM16A channel regulation by PI(4,5)P₂, demonstrating that PI(4,5)P₂-dependent TMEM16A channel activation can be allosterically modulated by phosphorylation and alternative splicing.