The effects of NMDA subunit composition on calcium influx and spike timing-dependent plasticity in striatal medium spiny neurons

Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development.     

Further characterization of regulation of Ca(V)2.2 by stargazin

Stargazin, a transmembrane protein expressed in the nervous system, shares similarities with the γ? subunit of skeletal muscle calcium channels. It was thus termed γ? subunit of neuronal calcium channels. Stargazin downregulates the expression of Ca(V)2 channels, however, its functional modulation of these channels remains debated. We have reported that Stargazin modulates Ca(V)2.2 channel by a Gβγ-dependent mechanism and suggested that Stargazin is not a true subunit of this channel, since all its effects on channel function are dependent on the presence of Gβγ. Moreover, Stargazin also modulated the GIRK channel in a Gβγ-dependent fashion. Here we report that Gβγ-dependent modulation by Stargazin of the biophysical properties of Ca(V)2.2 is unrelated to its negative effect on channel expression and current amplitude. Finally, we suggest that this Gβγ dependent modulation of Stargazin may have physiological relevance, since it was still present when we used Ca2(+) as charge carrier, instead of Ba2(+).     

Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels

Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.     

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation

It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.     

Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.

To cast light on the subunit composition of native NMDA-type glutamate receptors, four cloned subunits of the NMDA receptor have been expressed, in pairs, in Xenopus oocytes, and their single-channel properties have been measured. The conductances of the channels, and their characteristic patterns of sublevel transitions, turn out to be useful diagnostic criteria for subunit composition. The NR1-NR2A and NR1-NR2B combinations (which have identical TM2 sequences) are very similar to each other. Both have 50 pS openings and brief 40 pS sublevels (in 1 mM external Ca2+), with similar mean lifetimes and frequencies. They also show close quantitative resemblance to the channels of hippocampal CA1 and dentate gyrus cells and of cerebellar granule cells, except that the NR1-NR2A combination has a lower glycine sensitivity than the native channels. In contrast, the NR1-NR2C combination produces a channel with 36 pS and 19 pS conductances of similar (brief) duration; these closely resemble the 38-18 pS channels that have previously been observed in large cerebellar neurons in culture (together with 50 pS channels).     

Permeation and interaction of divalent cations in calcium channels of snail neurons

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.     

Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).     

Mutations in the EF-hand motif impair the inactivation of barium currents of the cardiac alpha1C channel.

Calcium-dependent inactivation has been described as a negative feedback mechanism for regulating voltage-dependent calcium influx in cardiac cells. Most recent evidence points to the C-terminus of the alpha1C subunit, with its EF-hand binding motif, as being critical in this process. The EF-hand binding motif is mostly conserved between the C-termini of six of the seven alpha1 subunit Ca2+ channel genes. The role of E1537 in the C-terminus of the alpha1C calcium channel inactivation was investigated here after expression in Xenopus laevis oocytes. Whole-cell currents were measured in the presence of 10 mM Ba2+ or 10 mM Ca2+ after intracellular injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Against all expectations, our results showed a significant reduction in the rate of voltage-dependent inactivation as measured in Ba2+ solutions for all E1537 mutants, whereas calcium-dependent inactivation appeared unscathed. Replacing the negatively charged glutamate residue by neutral glutamine, glycine, serine, or alanine significantly reduced the rate of Ba2+-dependent inactivation by 1.5-fold (glutamine) to 3.5-fold (alanine). The overall rate of macroscopic inactivation measured in Ca2+ solutions was also reduced, although a careful examination of the distribution of the fast and slow time constants suggests that only the slow time constant was significantly reduced in the mutant channels. The fast time constant, the hallmark of Ca2+-dependent inactivation, remained remarkably constant among wild-type and mutant channels. Moreover, inactivation of E1537A channels, in both Ca2+ and Ba2+ solutions, appeared to decrease with membrane depolarization, whereas inactivation of wild-type channels became faster with positive voltages. All together, our results showed that E1537 mutations impaired voltage-dependent inactivation and suggest that the proximal part of the C-terminus may play a role in voltage-dependent inactivation in L-type alpha1C channels.     

Positive regulation of capacitative Ca2+ entry by intracellular Ca2+ in Xenopus oocytes expressing rat TRP4

We have investigated the role of intracellular Ca2+ in the opening of capacitative Ca2+ entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl- current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl- response was recorded at controlled cytosolic Ca2+ concentrations. Intracellular perfusion with a Ca2+ free solution containing 10 mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+ concentration up to 300 nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+ concentration of 110 nM. Blockade of endogenous Cl- channels with anion channel blocker isolated Ca2+ current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+ concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+ at higher sensitivity compared to oocyte-endogenous CCE channels.     

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design.     

Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)- gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single- channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single- ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.     

Functional voltage-gated Ca2+ channels in muscle fibers of the platyhelminth Dugesia tigrina

The presence and function of voltage-gated Ca(2+) channels were examined in individual muscle fibers freshly dispersed from the triclad turbellarian Dugesia tigrina. Individual muscle fibers contracted in response to elevated extracellular K(+) in a concentration-dependent fashion. These depolarization-induced contractions were blocked by extracellular Co(2+) (2.5 mM), suggesting that they were dependent on depolarization-induced Ca(2+) influx across the sarcolemma. A voltage-gated inward current was apparent in whole cell recordings when the outward K(+) current was abolished by replacement of intracellular K(+) by Cs(+). This inward current was amplified with increasing concentration (相似文献   

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.     

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, IC, was activated by perfusing naive oocytes with a divalent-free recording solution. Ethanol (25-100 mM) modestly inhibited IC with 100 mM ethanol producing a 7-8% inhibition of steady state currents. The store-operated or capacitative calcium current (I(SOC)) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10-100 mM) dose-dependently inhibited the calcium-dependent chloride current (I(Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I(Cl(Ca)), I(Cl-1) and I(Cl-2). Under these conditions, ethanol (100 mM) inhibited I(Cl-1) currents to a greater extent (38%) than it did I(Cl-2) currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Regulation of the fertilization current in ascidian oocytes by intracellular second messengers

Neomycin, injected into ascidian oocytes to a final concentration of 10–50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm-activated fertilization currents are not inhibited in the presence of 100 μg/ml intracellular heparin, suggesting that these currents are not directly gated by InsP₃. The sulfhydryl reagent thimerosal at 100 μM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP₃ receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores. © 1994 Wiley-Liss, Inc.     

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.