Targeted disruption of the Ca2+ channel β3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca²⁺ channels, the pore-forming, antagonist-binding α₁ subunit, considerably less is understood about how β subunits contribute to neuronal Ca²⁺ channel function. We studied the role of the Ca²⁺ channel β₃ subunit, the major Ca²⁺ channel β subunit in neurons, by using a gene-targeting strategy. The β₃ deficient (β₃−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β₃−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca²⁺ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β₃ subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β₃−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca²⁺ channels. The reduction in the expression of N-type Ca²⁺ channels in the β₃−/− mice may be expected to impair Ca²⁺ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca²⁺ concentration and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. Herein we show that inhibitors of the Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca²⁺ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.     

A C-terminal motif found in the β2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins

The Na⁺/H⁺ exchanger regulatory factor (NHERF) binds to the tail of the β₂-adrenergic receptor and plays a role in adrenergic regulation of Na⁺/H⁺ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the β₂ receptor. Mutagenesis studies of the β₂ receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the β₂ receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.     

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na⁺/glucose cotransporter. The Na⁺/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na⁺/Cl⁻/γ-aminobutyric acid, Na⁺/iodide and H⁺/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.     

Ryanodine sensitizes the cardiac Ca2+ release channel (ryanodine receptor isoform 2) to Ca2+ activation and dissociates as the channel is closed by Ca2+ depletion

In single-channel recordings, the rabbit cardiac Ca(2+) release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by micromolar concentrations of ryanodine. At +30 mV, corresponding to a luminal to cytoplasmic cation current, the probability of opening (P(o)) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = -log(10) Ca concentration). However, at -30 mV, the P(o) was highly sensitive to Ca(2+) added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC(50) value for channel opening was about pCa 8. No significant Ca(2+) inactivation was observed for ryanodine-modified channels at either -30 mV or +30 mV. The opening of unmodified Ca(2+) channels is Ca(2+) sensitive, with an EC(50) value of about pCa 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC(50) values of pCa 2.2 at -30 mV and 2.5 at +30 mV. Mg(2+) decreased the P(o) of ryanodine-modified channels at low Ca(2+) concentrations at both -30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the P(o) of ryanodine-modified channels. In a [(3)H]ryanodine binding assay, [(3)H]ryanodine dissociation from the high-affinity binding site was found to be Ca(2+) sensitive, with an IC(50) of pCa 7.1. High concentrations of unlabeled ryanodine prevented [(3)H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca(2+) activation of the Ca(2+) release channel and desensitizes Ca(2+) inactivation through an allosteric interaction. [(3)H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca(2+), implying that high-affinity ryanodine and Ca(2+) binding sites are linked through either short- or long-range interactions, probably involving conformational changes.     

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na⁺- and K⁺-dependent pH homeostasis and in Na⁺ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H⁺, Na⁺/H⁺, and K⁺/H⁺ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H⁺ and Na⁺/H⁺ antiports is presented. K⁺ and Li⁺ inhibit Na⁺ uptake, but there is little cross-inhibition between Na⁺ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.     

Characterization of the thyroid Na+/I− symporter with an anti-COOH terminus antibody

The Na⁺/I⁻ symporter (NIS) is the plasma membrane protein that catalyzes active I⁻ transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; ≈65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a ≈87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the ≈87 kDa-polypeptide into a ≈50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (≈56 kDa) and the mature ≈87 kDa form. Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression in vivo was demonstrated.     

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the α-subunit of the human cardiac Na+ channel

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na⁺ current (I_Na,α) in human embryonic kidney (HEK293t) cells transfected with the α-subunit of the human cardiac Na⁺ channel (hH1_α). Extracellular application of 0.01 to 30 μM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I_Na,α with an IC₅₀ of 0.51 ± 0.06 μM. The EPA-induced suppression of I_Na,α was concentration- and voltage-dependent. EPA at 5 μM significantly shifted the steady-state inactivation relationship by −27.8 ± 1.2 mV (n = 6, P < 0.0001) at the V_1/2 point. In addition, EPA blocked I_Na,α with a higher “binding affinity” to hH1_α channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 μM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 μM EPA, from 2.1 ± 0.8 ms for control to 34.8 ± 2.1 ms (n = 5, P < 0.001). The effects of EPA on I_Na,α were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), α-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 μM and all-trans-retinoic acid at 10 μM had similar effects on I_Na,α as EPA. Even 5 μM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited I_Na,α. In contrast, 5 μM EPA ethyl ester did not alter I_Na,α (8 ± 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress I_Na,α with high “binding affinity” to hH1_α channels in the inactivated state and prolong the duration of recovery from inactivation.     

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP₃) binding to its receptors (IP₃R) in the endoplasmic reticulum (ER) activates Ca²⁺ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca²⁺ concentration signals including temporal oscillations and propagating waves. IP₃-mediated Ca²⁺ release is also controlled by cytoplasmic Ca²⁺ concentration with both positive and negative feedback. Single-channel properties of the IP₃R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP₃R on cytoplasmic Ca²⁺ and IP₃ concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP₃, even as [Ca²⁺]_i was raised to high concentrations, displaying two distinct types of functional Ca²⁺ binding sites: activating sites with half-maximal activating [Ca²⁺]_i (K_act) of 210 nM and Hill coefficient (H_act) ≈2; and inhibitory sites with half-maximal inhibitory [Ca²⁺]_i (K_inh) of 54 μM and Hill coefficient (H_inh) ≈4. Lowering IP₃ concentration was without effect on Ca²⁺ activation parameters or H_inh, but decreased K_inh with a functional half-maximal activating IP₃ concentration (K_IP3) of 50 nM and Hill coefficient (H_IP3) of 4 for IP₃. These results demonstrate that Ca²⁺ is a true receptor agonist, whereas the sole function of IP₃ is to relieve Ca²⁺ inhibition of IP₃R. Allosteric tuning of Ca²⁺ inhibition by IP₃ enables the individual IP₃R Ca²⁺ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca²⁺ concentration signaling and quantal Ca²⁺ release.     

Stress signaling through Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates salt adaptation in plants

Calcineurin (CaN) is a Ca²⁺- and calmodulin-dependent protein phosphatase (PP2B) that, in yeast, is an integral intermediate of a salt-stress signal transduction pathway that effects NaCl tolerance through the regulation of Na⁺ influx and efflux. A truncated form of the catalytic subunit and the regulatory subunit of yeast CaN were coexpressed in transgenic tobacco plants to reconstitute a constitutively activated phosphatase in vivo. Several different transgenic lines that expressed activated CaN also exhibited substantial NaCl tolerance, and this trait was linked to the genetic inheritance of the CaN transgenes. Enhanced capacity of plants expressing CaN to survive NaCl shock was similar when evaluation was conducted on seedlings in tissue culture raft vessels or plants in hydroponic culture that were transpiring actively. Root growth was less perturbed than shoot growth by NaCl in plants expressing CaN. Also, NaCl stress survival of control shoots was enhanced substantially when grafted onto roots of plants expressing CaN, further implicating a significant function of the phosphatase in the preservation of root integrity during salt shock. Together, these results indicate that in plants, like in yeast, a Ca²⁺- and calmodulin-dependent CaN signal pathway regulates determinants of salt tolerance required for stress adaptation. Furthermore, modulation of this pathway by expression of an activated regulatory intermediate substantially enhanced salt tolerance.     

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats

BACKGROUND & AIMS: Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS: Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS: Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS: In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.     

Differential roles of Ca2+/calmodulin-dependent kinases in posttetanic potentiation at input selective glutamatergic pathways

The electrosensory lateral line lobe (ELL) of the electric fish Apteronotus leptorhynchus is a layered medullary region receiving electroreceptor input that terminates on basal dendrites of interneurons and projection (pyramidal) cells. The molecular layer of the ELL contains two distinct glutamatergic feedback pathways that terminate on the proximal (ventral molecular layer, VML) and distal (dorsal molecular layer) apical dendrites of pyramidal cells. Western blot analysis with an antibody directed against mammalian Ca²⁺/calmodulin-dependent kinase 2, α subunit (CaMK2α) recognized a protein of identical size in the brain of A. leptorhynchus. Immunohistochemistry demonstrated that CaMK2 α expression in the ELL was restricted to fibers and terminals in the VML. Posttetanic potentiation (PTP) could be readily elicited in pyramidal cells by stimulation of either VML or DML in brain slices of the ELL. PTP in the VML was blocked by extracellular application of a CaMK2 antagonist (KN62) while intracellular application of KN62 or a CaMK2 inhibitory peptide had no effect, consistent with the presynaptic localization of CaMK2 α in VML. PTP in the dorsal molecular layer was not affected by extracellular application of KN62. Anti-Hebbian plasticity has also been demonstrated in the VML, but was not affected by KN62. These results demonstrate that, while PTP can occur independent of CaMK2, it is, in some synapses, dependent on this kinase.     

Syntaxin 1 interacts with the LD subtype of voltage-gated Ca2+ channels in pancreatic β cells

Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.     

瘦素在肝纤维化组织中的表达及其与肝星状细胞活化的关系

目的:探讨瘦素(leptin)在肝纤维化组织中的表达及其与肝纤维化过程中转化生长因子(TGF-β1)以及α平滑肌肌动蛋白(α-SMA)表达的相关性,了解leptin与肝星状细胞(HSC)活化的关系.方法:健康 SD大鼠40R,随机分成正常对照组和CCl4诱导的肝纤维化模型组.于造模2、4、6 wk末分批处死动物,分别采用RT-PCR和Western blot、免疫组织化学方法联合检测leptin、TGF-α1及α-SMA在肝纤维化组织中的表达.结果:正常对照组肝脏组织中leptin、TGF-β1、α-SMA均有微量表达;CCl4注射2wk后,leptin、TGF-β1、α-SMA表达开始增强,2、4、6 wk肝组织中的表达强度呈明显递增趋势(P<0.05).leptin与TGF-β1和α-SMA表达均呈显著相关性(r=0.668,0.570,均P<0.05).结论:leptin的阳性表达随着肝纤维化程度的加重而增强.在肝纤维化过程中leptin可能参与了HSC活化、增殖以及ECM的合成.     

肝星状细胞在逆转肝纤维化中的作用

肝纤维化是一种能够导致门静脉高压、肝硬化、肝衰竭的严重疾病。已经发现肝星状细胞的活化是引起肝纤维化的中心环节,因此抑制肝星状细胞的活化、加速肝星状细胞的清除有望逆转肝纤维化。本文将对活化的肝星状细胞的凋亡、衰老以及清除的研究进展作综述,阐明肝星状细胞在肝纤维化过程中所起的作用及相关机制。     

高血糖对大鼠肝纤维化及肝星状细胞活化的影响

目的探讨高血糖对大鼠肝纤维化及肝星状细胞活化的影响。方法取大鼠66只,随机分为4组,A组和B组均采用STZ(链脲佐菌素)和CCl4(四氯化碳)诱导为肝纤维化并糖尿病模型。A组诱导成功不做处理。B组糖尿病诊断成立后,皮下注射门冬胰岛素,3次/d控制血糖。C组单纯利用CCl4(四氯化碳分析纯)诱导为肝纤维化模型。D组(空白对照组)常规喂养,不做处理。观察各组大鼠一般情况(包括鼠食消耗、毛发变化、活动指数),8周后处死,测量大鼠体质量、肝脏体积及重量,采集血液及肝脏标本,分析对比各组大鼠肝脏系数,HE染色后光镜下肝组织纤维化情况;免疫组化SP法检测肝组织内α-SMA表达。结果实验各组与对照组大鼠相比体质量明显减轻(P<0.05),肝脏系数明显增加(P<0.05),肝组织α-SMA表达均不同程度增强(P<0.05);其中A组的体质量减轻最明显,肝纤维化、肝脏系数增加最显著(P<0.05),肝组织α-SMA表达亦有显著性差异(P<0.05);B组与C组相比体质量无明显减轻(P>0.05),肝脏系数无明显增加(P>0.05),而肝组织α-SMA表达差异有统计学意义(P<0.05)。结论高血糖可促进大鼠肝纤维化形成及肝星状细胞的活化。     

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca²⁺ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca²⁺ influx. To determine the Ca²⁺ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K⁺, Na⁺, or Ca²⁺. For modest concentrations of a single permeant cation, Ca²⁺ carried much more receptor current than did either K⁺ or Na⁺. For higher cation concentrations, however, the flux of Na⁺ or K⁺ through the transduction channel exceeded that of Ca²⁺. For mixtures of Ca²⁺ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca²⁺ over monovalent cations but also that Ca²⁺ carries substantial current even at low Ca²⁺ concentrations. At physiological cation concentrations, Ca²⁺ flux through transduction channels can change the local Ca²⁺ concentration in stereocilia in a range relevant for the control of adaptation.     

Ca2+-dependent and -independent interactions of the isoforms of the α1A subunit of brain Ca2+ channels with presynaptic SNARE proteins

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α_1A subunit of P/Q-type Ca²⁺ channels have different patterns of interactions with synaptic proteins. The BI isoform of α_1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca²⁺ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α_1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α_1A is Ca²⁺-dependent, with maximum affinity at 10–20 μM Ca²⁺. Although the rbA isoform of α_1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca²⁺-dependent. These differential, Ca²⁺-dependent interactions of Ca²⁺ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca²⁺ influx through these channels.