ApoE4-dependent lysosomal cholesterol accumulation impairs mitochondrial homeostasis and oxidative phosphorylation in human astrocytes.

Recent developments in genome sequencing have expanded the knowledge of genetic factors associated with late-onset Alzheimer's disease (AD). Among them, genetic variant ε4 of the APOE gene (APOE4) confers the greatest disease risk. Dysregulated glucose metabolism is an early pathological feature of AD. Using isogenic ApoE3 and ApoE4 astrocytes derived from human induced pluripotent stem cells, we find that ApoE4 increases glycolytic activity but impairs mitochondrial respiration in astrocytes. Ultrastructural and autophagy flux analyses show that ApoE4-induced cholesterol accumulation impairs lysosome-dependent removal of damaged mitochondria. Acute treatment with cholesterol-depleting agents restores autophagic activity, mitochondrial dynamics, and associated proteomes, and extended treatment rescues mitochondrial respiration in ApoE4 astrocytes. Taken together, our study provides a direct link between ApoE4-induced lysosomal cholesterol accumulation and abnormal oxidative phosphorylation.

Two-step structural changes in M3 muscarinic receptor activation rely on the coupled Gq protein cycle.

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the sequential activation-deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a Förster resonance energy transfer (FRET) tool for human M3 muscarinic receptor (hM3R), we find that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results reveal that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gßγ subunits. We also find that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cß. In sum, the present study uncovers the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle.

Reversibility and developmental neuropathology of linear nevus sebaceous syndrome caused by dysregulation of the RAS pathway.

Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous disorder caused by somatic gain-of-function mutations in KRAS or HRAS. LNSS brains have neurodevelopmental defects, including cerebral defects and epilepsy; however, its pathological mechanism and potentials for treatment are largely unclear. We show that introduction of KRASG12V in the developing mouse cortex results in subcortical nodular heterotopia and enhanced excitability, recapitulating major pathological manifestations of LNSS. Moreover, we show that decreased firing frequency of inhibitory neurons without KRASG12V expression leads to disrupted excitation and inhibition balance. Transcriptional profiling after destabilization domain-mediated clearance of KRASG12V in human neural progenitors and differentiating neurons identifies reversible functional networks underlying LNSS. Neurons expressing KRASG12V show molecular changes associated with delayed neuronal maturation, most of which are restored by KRASG12V clearance. These findings provide insights into the molecular networks underlying the reversibility of some of the neuropathologies observed in LNSS caused by dysregulation of the RAS pathway.

Dual regulation of Kv7.2/7.3 channels by long-chain n-alcohols.

Normal alcohols (n-alcohols) can induce anesthetic effects by acting on neuronal ion channels. Recent studies have revealed the effects of n-alcohols on various ion channels; however, the underlying molecular mechanisms remain unclear. Here, we provide evidence that long-chain n-alcohols have dual effects on Kv7.2/7.3 channels, resulting in channel activation as the net effect. Using heterologous expression systems, we found that n-alcohols could differentially regulate the Kv7.2/7.3 channel depending on their chain length. Treatment with short-chain ethanol and propanol diminished Kv7.2/7.3 currents, whereas treatment with long-chain hexanol and octanol enhanced the currents. However, the long-chain alcohols failed to potentiate Kv7.2 currents pre-activated by retigabine. Instead, they inhibited the currents, similar to short-chain ethanol. The stimulatory effect of the long-chain n-alcohols was also converted into an inhibitory one in the mutant Kv7.2(W236L) channels, while the inhibitory effect of ethanol did not differ between wild-type Kv7.2 and mutant Kv7.2(W236L). The inhibition of currents by n-alcohols was also seen in Kv7.1 channel which does not have the tryptophan (W) residue in S5. These findings suggest that long-chain n-alcohols exhibit dual effects through independent working sites on the Kv7.2 channel. Finally, we confirmed that the hydroxyl group with a negative electrostatic potential surface is essential for the dual actions of n-alcohol. Together, our data suggest that long-chain n-alcohols regulate Kv7.2/7.3 channels by interacting with both stimulatory and inhibitory sites and that their stimulatory action depends on the conserved tryptophan 236 residue in S5 and could be important for triggering their anesthetic effects.

Molecular basis of the PIP2-dependent regulation of CaV2.2 channel and its modulation by CaV ß subunits.

High-voltage-activated Ca2+ (CaV) channels that adjust Ca2+ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in an auxiliary CaV ß subunit-dependent manner. However, the molecular mechanism by which the ß subunits control the PIP2 sensitivity of CaV channels remains unclear. By engineering various α1B and ß constructs in tsA-201 cells, we reported that at least two PIP2-binding sites, including the polybasic residues at the C-terminal end of I-II loop and the binding pocket in S4II domain, exist in the CaV2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled CaV ß2 subunits. The membrane-anchored ß subunit abolished the PIP2 interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP2 sensitivity of CaV2.2 channels. By contrast, PIP2 interacted with the basic residues in the S4II domain of CaV2.2 channels regardless of ß2 isotype. Our data demonstrated that the anchoring properties of CaV ß2 subunits to the plasma membrane determine the biophysical states of CaV2.2 channels by regulating PIP2 coupling to the nonspecific phospholipid-binding site in the I-II loop.

OsSNDP3 Functions for the Polar Tip Growth in Rice Pollen Together with OsSNDP2, a Paralog of OsSNDP3.

Understanding pollen tube growth is critical for crop yield maintenance. The pollen tube provides a path for sperm cells for fertilization with egg cells. Cells must be subdivided into functionally and structurally distinct compartments for polar tip growth, and phosphoinositides are thought to be one of the facilitators for polarization during pollen tube growth. OsSNDP3 encodes Sec14-nodulin domain-containing protein and localizes in the nucleus and the microdomains of the plasma membrane in tobacco leaf epidermis cells. OsSNDP3 is thought to bind with phosphatidylinositol 4,5-bisphosphate based on the data including the information of basic amino acids in the C-terminal and colocalization with 2X Pleckstrin homology domain of Phospholipase C delta-1. OsSNDP3 interacts with a protein that contains a class I nodulin domain. We discovered that OsSNDP3 plays a significant role in pollen tube germination using CRISPR/Cas9 systems, whereas another pollen-preferential Sec14-nodulin domain-containing protein, OsSNDP2, additively functions with OsSNDP3 during pollen tube germination. Gene Ontology analysis using downregulated genes in ossndp3 indicated that the expression of genes involved in the phosphatidylinositol metabolic process and tip growth was significantly altered in ossndp3. OsSNDP3 aids pollen polar tip growth by binding with phosphatidylinositol 4,5-bisphosphate. We can better understand the roles of phosphoinositides during pollen tube growth by studying the functions of OsSNDP3 and OsSNDP2. And downregulated genes in ossndp3 might be useful targets for future research on polar tip growth.

Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells.

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

Posttranscriptional modulation of KCNQ2 gene expression by the miR-106b microRNA family.

MicroRNAs (miRNAs) have recently emerged as important regulators of ion channel expression. We show here that select miR-106b family members repress the expression of the KCNQ2 K+ channel protein by binding to the 3'-untranslated region of KCNQ2 messenger RNA. During the first few weeks after birth, the expression of miR-106b family members rapidly decreases, whereas KCNQ2 protein level inversely increases. Overexpression of miR-106b mimics resulted in a reduction in KCNQ2 protein levels. Conversely, KCNQ2 levels were up-regulated in neurons transfected with antisense miRNA inhibitors. By constructing more specific and stable forms of miR-106b controlling systems, we further confirmed that overexpression of precursor-miR-106b-5p led to a decrease in KCNQ current density and an increase in firing frequency of hippocampal neurons, while tough decoy miR-106b-5p dramatically increased current density and decreased neuronal excitability. These results unmask a regulatory mechanism of KCNQ2 channel expression in early postnatal development and hint at a role for miR-106b up-regulation in the pathophysiology of epilepsy.

Proprioception, the regulator of motor function.

In animals, proper locomotion is crucial to find mates and foods and avoid predators or dangers. Multiple sensory systems detect external and internal cues and integrate them to modulate motor outputs. Proprioception is the internal sense of body position, and proprioceptive control of locomotion is essential to generate and maintain precise patterns of movement or gaits. This proprioceptive feedback system is conserved in many animal species and is mediated by stretch-sensitive receptors called proprioceptors. Recent studies have identified multiple proprioceptive neurons and proprioceptors and their roles in the locomotion of various model organisms. In this review we describe molecular and neuronal mechanisms underlying proprioceptive feedback systems in C. elegans, Drosophila, and mice. [BMB Reports 2021; 54(8): 393-402].

Differential Regulation of Ca2+-Activated Cl- Channel TMEM16A Splice Variants by Membrane PI(4,5)P2.

TMEM16A is a Ca2+-activated Cl- channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP2 in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl- currents, which are independent of the membrane potential and specific to PI(4,5)P2 depletion. The attenuation of endogenous PI(4,5)P2 levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl- currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P2, not PI4P and PI(3,4,5)P3. Finally, we also confirmed that PI(4,5)P2 resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P2 selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.

Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains.

Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit.

Ethanol often causes critical health problems by altering the neuronal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P2), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K+ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (PO) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P2 depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P2 sensitivity of Kv7.2/7.3 channels. [BMB Reports 2021; 54(6): 311-316].

Phosphatidylinositol 4,5-bisphosphate is regenerated by speeding of the PI 4-kinase pathway during long PLC activation.

The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.

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 (PO). 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.

Ethanol Elevates Excitability of Superior Cervical Ganglion Neurons by Inhibiting Kv7 Channels in a Cell Type-Specific and PI(4,5)P2-Dependent Manner.

Alcohol causes diverse acute and chronic symptoms that often lead to critical health problems. Exposure to ethanol alters the activities of sympathetic neurons that control the muscles, eyes, and blood vessels in the brain. Although recent studies have revealed the cellular targets of ethanol, such as ion channels, the molecular mechanism by which alcohol modulates the excitability of sympathetic neurons has not been determined. Here, we demonstrated that ethanol increased the discharge of membrane potentials in sympathetic neurons by inhibiting the M-type or Kv7 channel consisting of the Kv7.2/7.3 subunits, which were involved in determining the membrane potential and excitability of neurons. Three types of sympathetic neurons, classified by their threshold of activation and firing patterns, displayed distinct sensitivities to ethanol, which were negatively correlated with the size of the Kv7 current that differs depending on the type of neuron. Using a heterologous expression system, we further revealed that the inhibitory effects of ethanol on Kv7.2/7.3 currents were facilitated or diminished by adjusting the amount of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). These results suggested that ethanol and PI(4,5)P2 modulated gating of the Kv7 channel in superior cervical ganglion neurons in an antagonistic manner, leading to regulation of the membrane potential and neuronal excitability, as well as the physiological functions mediated by sympathetic neurons.

Rapid resensitization of ASIC2a is conferred by three amino acid residues in the N terminus.

Acid-sensing ion channels (ASICs), sensory molecules that continuously monitor the concentration of extracellular protons and initiate diverse intracellular responses through an influx of cations, are assembled from six subtypes that can differentially combine to form various trimeric channel complexes and elicit unique electrophysiological responses. For instance, homomeric ASIC1a channels have been shown to exhibit prolonged desensitization, and acid-evoked currents become smaller when the channels are repeatedly activated by extracellular protons, whereas homomeric or heteromeric ASIC2a channels continue to respond to repetitive acidic stimuli without exhibiting such desensitization. Although previous studies have provided evidence that both the desensitization of ASIC1a and rapid resensitization of ASIC2a commonly require domains that include the N terminus and the first transmembrane region of these channels, the biophysical basis of channel gating at the amino acid level has not been clearly determined. Here, we confirm that domain-swapping mutations replacing the N terminus of ASIC2a with that of ASIC2b result in de novo prolonged desensitization in homomeric channels following activation by extracellular protons. Such desensitization of chimeric ASIC2a mutants is due neither to internalization nor to degradation of the channel proteins. We use site-directed mutagenesis to narrow down the relevant portion of the N terminus of ASIC2a, identifying three amino acid residues within the N terminus (T25, T39, and I40) whose mutation is sufficient to phenocopy the desensitization exhibited by the chimeric mutants. A similar desensitization is observed in heteromeric ASICs containing the mutant subunit. These results suggest that T25, T39, and I40 of ASIC2a are key residues determining the rapid resensitization of homomeric and heteromeric ASIC2a channels upon proton activation.

Translocatable voltage-gated Ca2+ channel ß subunits in α1-ß complexes reveal competitive replacement yet no spontaneous dissociation.

ß subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-ß interactions by rapamycin-translocatable CaV ß subunits that allow drug-induced sequestration and uncoupling of the ß subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified ß subunits, except membrane-tethered ß2a and ß2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable ß subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound ß subunits are very stable. However, the interaction becomes dynamic when other competing ß isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free ß isoforms around the channel reveals a dynamic aspect to the α1B-ß interaction. On the other hand, translocatable ß subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable ß subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-ß subunits can be overcome by other free ß isoforms, permitting dynamic changes in channel properties in intact cells.

The HOOK region of ß subunits controls gating of voltage-gated Ca2+ channels by electrostatically interacting with plasma membrane.

Recently, we showed that the HOOK region of the ß2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity of voltage-gated Ca2+ (CaV) 2.2 channels. Here, we report that voltage-dependent gating and current density of the CaV2.2 channels are also regulated by the HOOK region of the ß2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of CaV2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the CaV2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the ß2 subunit plays an important role in determining the overall CaV channel gating properties.

The HOOK region of voltage-gated Ca2+ channel ß subunits senses and transmits PIP2 signals to the gate.

The ß subunit of voltage-gated Ca2+ (CaV) channels plays an important role in regulating gating of the α1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular localization of the CaV ß subunit is critical for this effect; N-terminal-dependent membrane targeting of the ß subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the ß subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a ß subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a ß subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The ß2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the ß subunit acts as an important regulator of CaV channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane.