The Ca2+ channel β2 subunit is selectively targeted to the axon terminals of supraoptic neurons

The assembly of high voltage-activated Ca²⁺ channels with different β subunits influences channel properties and possibly subcellular targeting. We studied β subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca_Vβ subunits (Ca_Vβ₁-Ca_Vβ₄) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 β subunits are expressed in both locations, but that Ca_Vβ₂ had the highest relative expression in the neurohypophysis. These data suggest that the Ca_Vβ₂ subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca²⁺ channels.     

Stargazin Modulates Neuronal Voltage-dependent Ca2+ Channel Cav2.2 by a Gβγ-dependent Mechanism

Loss of neuronal protein stargazin (γ₂) is associated with recurrent epileptic seizures and ataxia in mice. Initially, due to homology to the skeletal muscle calcium channel γ₁ subunit, stargazin and other family members (γ_3–8) were classified as γ subunits of neuronal voltage-gated calcium channels (such as Ca_V2.1-Ca_V2.3). Here, we report that stargazin interferes with G protein modulation of Ca_V2.2 (N-type) channels expressed in Xenopus oocytes. Stargazin counteracted the Gβγ-induced inhibition of Ca_V2.2 channel currents, caused either by coexpression of the Gβγ dimer or by activation of a G protein-coupled receptor. Expression of high doses of Gβγ overcame the effects of stargazin. High affinity Gβγ scavenger proteins m-cβARK and m-phosducin produced effects similar to stargazin. The effects of stargazin and m-cβARK were not additive, suggesting a common mechanism of action, and generally independent of the presence of the Ca_Vβ₃ subunit. However, in some cases, coexpression of Ca_Vβ₃ blunted the modulation by stargazin. Finally, the Gβγ-opposing action of stargazin was not unique to Ca_V2.2, as stargazin also inhibited the Gβγ-mediated activation of the G protein-activated K⁺ channel. Purified cytosolic C-terminal part of stargazin bound Gβγ in vitro. Our results suggest that the regulation by stargazin of biophysical properties of Ca_V2.2 are not exerted by direct modulation of the channel but via a Gβγ-dependent mechanism.     

Single-Channel Kinetics,Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP₃R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K⁺ as the charge carrier (cytoplasmic [IP₃] = 10 μM, free [Ca²⁺] = 200 nM), the IP₃R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean P _o = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F₁ and F₂. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F₁ to F₂ transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP₃ and the absence of change in [Ca²⁺]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP₃R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca²⁺-independent inactivation of IP₃R and channel clustering may contribute to complex [Ca²⁺] signals in cells.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes: Antagonistic effects of Gβγ and protein kinase C

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K⁺ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca²⁺ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 3₁₀-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K⁺ channel but is not capable of activating skeletal-type Ca²⁺ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K⁺ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K⁺ channel inhibitors and Ca²⁺ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K⁺ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.     

The α2δ subunit augments functional expression and modifies the pharmacology of CaV1.3 L-type channels

The auxiliary Ca_Vα₂δ-1 subunit is an important component of voltage-gated Ca²⁺ (Ca_V) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca_V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca_Vα₂δ-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca_V1.3 channels in HEK-293 cells. Co-expression of Ca_Vα₂δ-1 significantly increased macroscopic currents and conferred the Ca_V1.3α₁/Ca_Vβ₃ channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca_Vα₂δ-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane δ domain of the protein, reduced functionality in terms of enhancement of Ca_V1.3α₁/Ca_Vβ₃ currents. In addition, co-expression of the δ domain drastically inhibited macroscopic currents through recombinant Ca_V1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca_Vα₂δ-1 auxiliary subunit may interact with Ca_V1.3 channels and regulate their functional expression.     

The smooth muscle-type β1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels

Large-conductance Ca²⁺-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC₄(3) does not depend, as initially proposed, on the β₁ or β₄ subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the β₁ subunit is not essential to this activation but exerts a large potentiating effect. DiBAC₄(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca²⁺. K_d values for BK channel activation by DiBAC₄(3) in 0 mM Ca²⁺ are approximately 20 μM (α) and 5 μM (α+β₁), and G-V curves shift up to −40mV and −110 mV, respectively. β₁ to β₂ mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the β₁ subunit in cardiovascular pharmacology.     

CaV1.2 I-II linker structure and Timothy syndrome

Ca_V channels are multi-subunit protein complexes that enable inward cellular Ca²⁺ currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker’s helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca_V1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant’s (human Ca_V1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.     

Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

Effects of internal Sr²⁺ on the activity of large-conductance Ca²⁺-activated K⁺ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr²⁺ was approximately one-fourth as potent as Ca²⁺ in activating these channels. Although the Hill coefficient for Sr²⁺ was smaller than that for Ca²⁺, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca²⁺ and Sr²⁺. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr²⁺ at higher concentrations (>100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr²⁺ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τ_l−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τ_b). A significant decrease in τ_b and no large changes in τ_l−s were observed with increased Sr²⁺ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr²⁺ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba²⁺ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).     

Regulation of Insulin Secretion in Islets of Langerhans by Ca<Superscript>2+</Superscript>Channels

Insulin secretion from β-cells of the pancreatic islets of Langerhans is triggered by Ca²⁺ influx through voltage-dependent Ca²⁺ channels. Electrophysiological and molecular studies indicate that β-cells express several subtypes of these channels. This review discusses their roles in regulating insulin secretion, focusing on recent studies using β-cells, exogenous expression systems, and Ca²⁺ channel knockout mice. These investigations reveal that L-type Ca²⁺ channels in the β-cell physically interact with the secretory apparatus by binding to synaptic proteins on the plasma membrane and insulin granule. As a result, Ca²⁺ influx through L-type channels efficiently and rapidly stimulates release of a pool of insulin granules in close contact with the channels. Thus, L-type Ca²⁺ channel activity is preferentially coupled to exocytosis in the β-cell, and plays a critical role in regulating the dynamics of insulin secretion. Non-L-type channels carry a significant portion of the total voltage-dependent Ca²⁺ current in β-cells and cell lines from some species, but nevertheless account for only a small fraction of insulin secretion. These channels may regulate exocytosis indirectly by affecting membrane potential or second messenger signaling pathways. Finally, voltage-independent Ca²⁺ entry pathways and their potential roles in β-cell function are discussed. The emerging picture is that Ca²⁺ channels regulate insulin secretion at multiple sites in the stimulus-secretion coupling pathway, with the specific role of each channel determined by its biophysical and structural properties.This revised version was published online in June 2005 with a corrected cover date.     

Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line

The stimulation of IP₃ production by muscarinic agonists causes both intracellular Ca²⁺ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na⁺ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na⁺ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na⁺ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent K _D of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca²⁺ influx and electrical excitability. In N1E-115 cells, Ca²⁺ entry by this pathway is necessary to refill the IP₃-sensitive intracellular Ca²⁺ pool during repeated stimulation and CNG channels may play a similar role in other neurons.     

Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca²⁺ homeostasis plays a key role in cardiac function, we investigated how Ca²⁺ channels and Ca²⁺ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca²⁺ imaging experiments proved the functional expression of the L-type Ca²⁺ current (I_Ca) starting from day 7 of EB development. On day 7, using 10 mM Ca²⁺ as charge carrier, I_Ca was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of I_Ca proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, I_Ca was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP₃-sensitive intracellular Ca²⁺ stores and Ca²⁺-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca²⁺ stores, detected by caffeine-induced Ca²⁺ release, appeared in most GFP-positive cells 1–2 d after I_Ca. Coexpression of both I_Ca and ryanodine-sensitive Ca²⁺ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.Thus, the functional expression of voltage-dependent L-type Ca²⁺ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP₃ receptors and sarcoplasmic Ca²⁺-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Structural Basis for Calcium and Magnesium Regulation of a Large Conductance Calcium-activated Potassium Channel with β1 Subunits

Large conductance Ca²⁺- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary β subunits, play important roles in diverse physiological activities. The β1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca²⁺ sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and β1 remains elusive. Using macroscopic ionic current recordings in various Ca²⁺ and Mg²⁺ concentrations, we identified two binding sites on the cytosolic N terminus of β1, namely the electrostatic enhancing site (mSlo1(K392,R393)-β1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-β1(L5,V6,M7)), passing the physical force from the Ca²⁺ bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg²⁺ sensitivity. A comprehensive structural model of the BK(mSlo1 α-β1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.     

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca²⁺ channel complexes. Ca_Vα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca_V1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca_Vα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca_Vα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca_Vα2δ1-mediated increase in the peak current density and voltage-dependent gating of Ca_V1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca_Vα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca_Vα2δ1 as well as the Ca_Vα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca_Vα2δ1, and furthermore that N-glycosylation of Ca_Vα2δ1 is essential to produce functional L-type Ca²⁺ channels.     

Alteration of channel characteristics by exchange of pore-forming regions between two structurally related Ca2+ Channels

Several types of structurally homologous high voltage-gated Ca²⁺ channels (L-, P-and N-type) have been identified via biochemical, pharmacological and electrophysiological techniques. Among these channels, the cardiac L-type and the brain BI-2 Ca²⁺ channel display significantly different biophysical properties. The BI-2 channel exhibits more rapid voltage-dependent current activation and inactivation and smaller single-channel conductance compared to the L-type Ca²⁺ channel. To examine the molecular basis for the functional differences between the two structurally related Ca²⁺ channels, we measured macroscopic and single-channel currents from oocytes injected with wild-type and various chimeric channel ₁ subunit cRNAs. The results show that a chimeric channel in which the segment between S5-SS2 in repeat IV of the cardiac L-type Ca²⁺ channel, was replaced by the corresponding region of the BI-2 channel, exhibited macroscopic current activation and inactivation time-courses and single-channel conductance, characteristic of the BI-2 Ca²⁺ channel. The voltage-dependence of steady-state inactivation was not affected by the replacement. Chimeras, in which the SS2-S6 segment in repeat III or IV of the cardiac channel was replaced by the corresponding BI-2 sequence, exhibited altered macroscopic current kinetics without changes in single-channel conductance. These results suggest that part of the S5-SS2 segment plays a critical role in determining voltage-dependent current activation and inactivation and single-channel conductance and that the SS2-S6 segment may control voltage-dependent kinetics of the Ca²⁺ channel.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

A Short Polybasic Segment between the Two Conserved Domains of the β2a-Subunit Modulates the Rate of Inactivation of R-type Calcium Channel

Besides opening and closing, high voltage-activated calcium channels transit to a nonconducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of a deleterious rise in calcium concentration. R-type high voltage-activated channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular β-subunit isoform, β_2a, inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N terminus anchors β_2a to the plasma membrane. The current view is that membrane-anchored β_2a immobilizes the channel inactivation machinery and confers slow inactivation phenotype. β-Subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here, we identified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient β_2a to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slow inactivation-conferring phenotype. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the β_2a-subunit that might involve electrostatic interactions with an unknown target on the channel''s inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the β_2a-subunit.     

Effects of Copper on T-Type Ca<Superscript>2+</Superscript> Channels in Mouse Spermatogenic Cells

Low voltage-activated, rapidly inactivating T-type Ca²⁺ channels are found in a variety of cells, where they regulate electrical activity and Ca²⁺ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu²⁺) inhibited T-type Ca²⁺ current (I _T-Ca) with IC₅₀ of 12.06 μM. Inhibition of I _T-Ca by Cu²⁺ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu²⁺ (10 μM) inhibited I _T-Ca by 49.6 ± 4.1%. Inhibition of I _T-Ca by Cu²⁺ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu²⁺ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca²⁺ channels, we analyzed the effect of Cu²⁺ on activation, inactivation, deactivation and reactivation of T-type Ca²⁺ channels. Since T-type Ca²⁺ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu²⁺ can affect male reproductive function through T-type Ca²⁺ channels as a preconception contraceptive material.