Inactivation of Gating Currents of L-Type Calcium Channels : Specific Role of the α2δ Subunit

In studies of gating currents of rabbit cardiac Ca channels expressed as α_1C/β_2a or α_1C/β_2a/α₂δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V_1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V_1/2 ≃ −90 mV) is generated in inactivated channels. In α_1C/β_2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α₂δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α₂δ subunit did not change the V_1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α₂δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode.     

BIN1 Localizes the L-Type Calcium Channel to Cardiac T-Tubules

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.     

Antigens Associated with N- and L-Type Calcium Channels in Lambert-Eaton Myasthenic Syndrome

Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (¹²⁵I-ω conotoxin GVIA) and L-type ([³H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.     

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release

Aberrant Zn²⁺ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn²⁺ modulates cardiac ryanodine receptor gating and Ca²⁺ dynamics in isolated cardiomyocytes. We reveal that Zn²⁺ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn²⁺ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca²⁺. At concentrations of free Zn²⁺ >1 nm, Zn²⁺ is the main activating ligand, and the dependence on Ca²⁺ is removed. Zn²⁺ is therefore a higher affinity activator of RyR2 than Ca²⁺. Millimolar levels of free Zn²⁺ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn²⁺ modulates both the frequency and amplitude of Ca²⁺ waves in a concentration-dependent manner and that physiological levels of Zn²⁺ elicit Ca²⁺ release in the absence of activating levels of cytosolic Ca²⁺. This highlights a new role for intracellular Zn²⁺ in shaping Ca²⁺ dynamics in cardiomyocytes through modulation of RyR2 gating.     

Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells

L-type calcium currents (I_Ca) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I_Ca and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca²⁺ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α₂δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region.     

Gating of Single N-type Calcium Channels Recorded from Bullfrog Sympathetic Neurons

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba²⁺ in the pipet, mean N-channel open probability (P _o, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., P _o at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (τ_o) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (τ_sh1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (τ_sh2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel P _o during individual sweeps at +40 mV was often high (∼0.8), mean P _o was reduced by null sweeps, low P _o gating, inactivation, and slow activation. The variability in mean P _o across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low P _o gating (P _o = 0.2, τ_o = 1 ms at +40 mV) could be sustained for ∼1 min in some patches. The clustering of null sweeps and sweeps with low P _o gating is consistent with the idea that they result from different modes of N-channel gating. While P _o of the main N-channel gating state is high, the net P _o is reduced to a maximum value of close to 0.5 by other gating processes.     

Glycosylation Influences Voltage-Dependent Gating of Cardiac and Skeletal Muscle Sodium Channels

The role of glycosylation on voltage-dependent channel gating for the cloned human cardiac sodium channel (hH1a) and the adult rat skeletal muscle isoform (μl) was investigated in HEK293 cells transiently transfected with either hH1a or μl cDNA. The contribution of sugar residues to channel gating was examined in transfected cells pretreated with various glycosidase and enzyme inhibitors to deglycosylate channel proteins. Pretreating transfected cells with enzyme inhibitors castanospermine and swainsonine, or exo-glycosidase neuroaminidase caused 7 to 9 mV depolarizing shifts of V _1/2 for steady-state activation of hH1a, while deglycosylation with corresponding drugs elicited about the same amount of depolarizing shifts (8 to 9 mV) of V _1/2 for steady-state activation of μl. Elevated concentrations of extracellular Mg²⁺ significantly masked the castanospermine-elicited depolarizing shifts of V _1/2 for steady-state activation in both transfected hH1a and μl. For steady-state activation, deglycosylation induced depolarizing shifts of V _1/2 for hH1a (10.6 to 12 mV), but hyperpolarizing shifts for μl (3.6 to 4.4 mV). Pretreatment with neuraminidase had no significant effects on single-channel conductance, the mean open time, and the open probability. These data suggest that glycosylation differentially regulates Na channel function in heart and skeletal muscle myocytes. Received: 8 April 1999/Revised: 18 June 1999     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Postsynaptic GABABRs Inhibit L-Type Calcium Channels and Abolish Long-Term Potentiation in Hippocampal Somatostatin Interneurons

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Actin Modulates the Gating of Neurospora crassa VDAC

VDAC forms the major pathway for metabolites across the mitochondrial outer membrane. The regulation of the gating of VDAC channels is an effective way to control the flow of metabolites into and out of mitochondria. Here we present evidence that actin can modulate the gating process of Neurospora crassa VDAC reconstituted into membranes made with phosphatidylcholine. An actin concentration as low as 50 nm caused the VDAC-mediated membrane conductance to drop by as much as 85% at elevated membrane potentials. Actin's effect could be quickly reversed by adding pronase to digest the protein. α-Actin, from mammalian muscle, has a stronger effect than β- and γ-actin from human platelets. The monomeric form of actin, G-actin, is effective. Stabilization of the fibrous form, F-actin, with the mushroom toxin, phalloidin, blocks the effect of actin on VDAC, indicating that F-actin might be ineffective. Cytochalasin B did not interfere with the ability of actin to favor VDAC closure. DNase-I did effectively block actin's effect on VDAC, and VDAC decreased actin's inhibitory effect on DNase-I activity, indicating that N. crassa VDAC competes with DNase-I for the same binding site on actin. The actin-VDAC interaction might be a mechanism by which actin regulates energy metabolism. Received: 28 August 2000/Revised: 1 December 2000     

A Long-Term Blockade of L-Type Calcium Currents Upregulates the Number of Ca2+ Channels in Skeletal Muscle

The effects of a long-term blockade of L-type Ca²⁺ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers. Ca²⁺ currents (I _Ca) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of I _Ca increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca²⁺ channel blocker nifedipine or in Ca²⁺-free conditions. A similar incubation period with Cd²⁺, an inorganic blocker, produced a moderate increase of 20% in peak I _Ca. The maximum mobilized charge (Q _max) increased by 50% in fibers preincubated in Ca²⁺-free solutions or in the presence of Cd²⁺. Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd²⁺ prior to the isolation of the microsomal fraction doubled the number of ³H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the number of binding sites is consistent with the increase in the peak amplitude of I _Ca as well as with the increase in Q _max. Received: 31 August 1998/Revised: 7 December 1998     

Mechanosensitive Gating of Kv Channels

K-selective voltage-gated channels (Kv) are multi-conformation bilayer-embedded proteins whose mechanosensitive (MS) P_open(V) implies that at least one conformational transition requires the restructuring of the channel-bilayer interface. Unlike Morris and colleagues, who attributed MS-Kv responses to a cooperative V-dependent closed-closed expansion↔compaction transition near the open state, Mackinnon and colleagues invoke expansion during a V-independent closed↔open transition. With increasing membrane tension, they suggest, the closed↔open equilibrium constant, L, can increase >100-fold, thereby taking steady-state P_open from 0→1; “exquisite sensitivity to small…mechanical perturbations”, they state, makes a Kv “as much a mechanosensitive…as…a voltage-dependent channel”. Devised to explain successive g_K(V) curves in excised patches where tension spontaneously increased until lysis, their L-based model falters in part because of an overlooked IK feature; with recovery from slow inactivation factored in, their g(V) datasets are fully explained by the earlier model (a MS V-dependent closed-closed transition, invariant L≥4). An L-based MS-Kv predicts neither known Kv time courses nor the distinctive MS responses of Kv-ILT. It predicts Kv densities (hence gating charge per V-sensor) several-fold different from established values. If opening depended on elevated tension (L-based model), standard g_K(V) operation would be compromised by animal cells’ membrane flaccidity. A MS V-dependent transition is, by contrast, unproblematic on all counts. Since these issues bear directly on recent findings that mechanically-modulated Kv channels subtly tune pain-related excitability in peripheral mechanoreceptor neurons we undertook excitability modeling (evoked action potentials). Kvs with MS V-dependent closed-closed transitions produce nuanced mechanically-modulated excitability whereas an L-based MS-Kv yields extreme, possibly excessive (physiologically-speaking) inhibition.     

Open-State Occupancy Prevents Gating Charge Relaxation of N-type (CaV2.2) Calcium Channels

N-type and L-type channels have significant gating differences, and we wondered whether some of these differences are linked to the relationship between charge movement and channel opening. The time constants for N-channel closing (τ_Deact) and Off-gating charge movement (τQ_Off) were compared over a range of voltages. τQ_Off was significantly larger than τ_Deact at voltages < −10 mV, and the voltage dependence of the τQ_Off was less steep than that for τ_Deact, which suggests that gating charge relaxation does not limit channel closing. Roscovitine, a drug that slows N-channel closing by holding the channel in a high open-probability state, was found to slow both τQ_Off and τ_Deact, and thus the time courses of channel closing and gating charge relaxation were similar. Our gating current results were reproduced with the addition of a voltage-independent, closed-closed transition to our previously published two-open-state N-channel model. This work suggests that, like L-type channels, there is a voltage-independent transition along the N-channel activation/deactivation pathway, but this transition occurs between closed states instead of the closed-open states of the L-channel. Also unlike L-type channels, the gating charge appears to be locked into the activated position by the N-channel open state.     

Protein Kinase C Modulates Calcium Channels in Isolated Presynaptic Nerve Terminals of Rat Hippocampus

Abstract: Nerve terminals (“synaptosomes”) isolated from rat brain hippocampus were loaded with the fluorescent Ca²⁺ indicator fura-2 and were subjected to depolarization with an elevated K⁺ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca²⁺ channels in the presynaptic membrane. Three components of Ca²⁺ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca²⁺ channels with different inactivation kinetics (τ of ～60 ms and 1 s, respectively) and Na⁺/Ca²⁺ exchange working in the “reverse” mode. The activity of both classes of voltage-dependent Ca²⁺ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca²⁺ channel activity, but the effect of DHS could be showed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca²⁺ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.     

Spontaneous Gating of Olfactory Cyclic-Nucleotide-Gated Channels

In vertebrates, cilia on the olfactory receptor neurons have a high density of cyclic-nucleotide-gated (CNG) channels. During transduction of odorous stimuli, cyclic AMP is formed. cAMP gates the CNG channels and this initiates the neuronal depolarization. Here it is shown that the ciliary CNG channels also open spontaneously. In the absence of odorants and second messengers, olfactory cilia have a small basal conductance to cations. Part of this conductance is similar to the cAMP-activated conductance in its sensitivity to channel inhibitors and divalent cations. The basal conductance may help to stabilize the neuronal membrane potential while limiting the sensitivity of odorant detection. Received: 30 May 2000/Revised: 8 August 2000     

Gating Transition of Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are an important family of membrane proteins and play key roles in physiological processes, including signal transduction at chemical synapses. Here, we study the conformational changes associated with the opening and closing of the channel pore. Based on recent crystal structures of two prokaryotic members of the family in open and closed states, respectively, mixed elastic network models are constructed for the transmembrane domain. To explore the conformational changes in the gating transition, a coarse-grained transition path is computed that smoothly connects the closed and open conformations of the channel. We find that the conformational transition involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.     

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is an important regulator of intracellular Ca²⁺ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na⁺/K⁺-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca²⁺ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X_5–8Φ₁Φ₂-X_8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.     

Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells

Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Ca_v). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Ca_v opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet β-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Ca_vα1 pore-forming subunits of R-type Ca_v2.3 and to lesser extent L-type Ca_vs, while confirming the preferential interactions between Syn-1A with L-type (Ca_v1.2, Ca_v1.3) Ca_vs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Ca_v2.3, whereas Syn-1A prefers L-type Ca_vs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Ca_v channels. Syn-3 KD enhanced Ca²⁺ currents by 46% attributed mostly to R- and L-type Ca_vs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Ca_v activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Ca_v activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Ca_vs, but preferring Ca_v2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS.     

Inorganic Polyphosphate Modulates TRPM8 Channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P₂). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.