QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (104) Citing Articles via Google Scholar Google Scholar Articles by Tottene, A. Articles by Pietrobon, D. Search for Related Content PubMed PubMed Citation Articles by Tottene, A. Articles by Pietrobon, D.

 Previous Article  |  Next Article 

The Journal of Neuroscience, January 1, 2000, 20(1):171-178

_1E Subunits Form the Pore of Three Cerebellar R-Type Calcium Channels with Different Pharmacological and Permeation Properties

Angelita Tottene¹, Stephen Volsen², and Daniela Pietrobon¹

¹ Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy, and ² Lilly Research Center, Eli Lilly Company Limited, Windlesham, Surrey GU20 6PH, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

R-type Ca²⁺ channels cooperate with P/Q- and N-type channels to control neurotransmitter release at central synapses. The leading candidate as pore-forming subunit of R-type channels is the _1E subunit. However, R-type Ca²⁺ currents with permeation and/or pharmacological properties different from those of recombinant Ca²⁺ channels containing _1E subunits have been described, and therefore the molecular nature of R-type Ca²⁺ channels remains not completely settled. Here, we show that the R-type Ca²⁺ current of rat cerebellar granule cells consists of two components inhibited with different affinity by the _1E selective antagonist SNX482 (IC₅₀ values of 6 and 81 nM) and a third component resistant to SNX482. The SNX482-sensitive R-type current shows the unique permeation properties of recombinant _1E channels; it is larger with Ca²⁺ than with Ba²⁺ as charge carrier, and it is highly sensitive to Ni²⁺ block and has a voltage-dependence of activation consistent with that of G2 channels with unitary conductance of 15 pS. On the other hand, the SNX482-resistant R-type current shows permeation properties similar to those of recombinant _1A and _1B channels; it is larger with Ba²⁺ than with Ca²⁺ as charge carrier_, and it has a low sensitivity to Ni²⁺ block and a voltage-dependence of activation consistent with that of G3 channels with unitary conductance of 20 pS. Gene-specific knock-down by antisense oligonucleotides demonstrates that the different cerebellar R-type channels are all encoded by the _1E gene, suggesting the existence of _1E isoforms with different pore properties.

Key words: calcium channel; _1E subunit; antisense oligonucleotides; cerebellum; granule cells; permeation; toxin-resistant calcium current

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Most neurons of the CNS display several types of high-voltage activated Ca²⁺ channels, pharmacologically classified as L-, N-, P/Q, and R-type (Dunlap et al., 1995). R-type Ca²⁺ currents were first described by Tsien and coworkers as corresponding to the residual current observed in rat cerebellar granule neurons after pharmacological block of L-, N-, and P/Q-type Ca²⁺ channels (Zhang et al., 1993; Randall and Tsien, 1995). Subsequently, R-type currents with different biophysical properties were described in different types of neurons (Tottene et al., 1996; Hilaire et al., 1997; Magnelli et al., 1998; Wu et al., 1998), and single channel recordings revealed that rat cerebellar granule cells coexpress two Ca²⁺ channels, called G2 and G3, differing in unitary conductance and threshold for activation but both classified as R-type on the basis of their resistance to all specific Ca²⁺ channel inhibitors (Forti et al., 1994; Tottene et al., 1996).

The molecular basis of R-type channels is not completely settled. The leading candidate as pore-forming subunit of R-type channels is the _1E subunit. However, of the two R-type channels of cerebellar granule cells, only G2 has a unitary conductance of 15 pS, similar to that of recombinant _1E channels (Schneider et al., 1994; Wakamori et al., 1994; Bourinet et al., 1996), whereas G3 has a conductance of 20 pS (Forti et al., 1994; Tottene et al., 1996). Whereas recombinant _1E channels have the unique property among high-voltage activated channels of carrying more current with Ca²⁺ than with Ba²⁺ (Bourinet et al., 1996)_, and in addition are very sensitive to Ni²⁺ block (Soong et al., 1993; Schneider et al., 1994; Williams et al., 1994; Zamponi et al., 1996), native R-type currents show Ba²⁺ over Ca²⁺ current ratios ranging from 0.9 to 2 (Zhang et al., 1993; Hilaire et al., 1997; Magnelli et al., 1998), and some require relatively high concentrations of Ni²⁺ to be blocked (Tottene et al., 1996; Magnelli et al., 1998; Wu et al., 1998). SNX482, the first selective antagonist of recombinant _1E channels, failed to inhibit R-type currents in several types of central neurons, including rat cerebellar granule cells (Newcomb et al., 1998). However, the R-type current of these neurons was reduced by antisense oligonucleotides (ONs) against _1E (Piedras-Renteria and Tsien, 1998).

Whereas splice variants of ₁ subunits with different pharmacological properties are known (Bourinet et al., 1999; Hans et al., 1999), there are to date no reports of splice variants or subunit combinations with different permeation properties and/or single channel conductance. Therefore, it remains unclear whether the different native R-type channels and in particular G2 and G3 are all encoded by the _1E gene. Indeed, it has been suggested that some R-type currents might actually be supported by channels containing _1A or _1B subunits with low affinity for their specific toxins (Nooney et al., 1997; Magnelli et al., 1998).

Using an antisense strategy, here we show that the _1E gene encodes both native SNX482-sensitive R-type channels with the unique permeation properties of recombinant _1E channels and native SNX482-resistant R-type channels showing none of these properties.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Cerebellar granule cells were grown in primary culture after enzymatic and mechanical dissociation from 6- to 7-d-old Wistar rats according to the procedure of Levi et al. (1984). The cells were plated on poly-L-lysine-coated glass coverslips and kept in Basal Eagle's medium supplemented with 10% fetal calf serum, 25 mM KCl, 2 mM glutamine, and 60 µg/ml gentamycin. Cytosine arabinoside (10 µM) was added to the culture 18 hr after plating to inhibit the proliferation of non-neuronal cells. Granule cells were the large majority of the cells in the cultures and were morphologically identified by their oval or round cell body, small size, and bipolar neurites. Experiments were usually performed on granule cells grown for 5-7 d in vitro.

Transfection with oligonucleotides. One day after plating, cerebellar granule cells were transfected using polyethylenimine (PEI) (50 kDa; Sigma, St. Louis, MO) as transfecting agent (Lambert et al., 1996). DNA (1 µg) and 60 nmol of PEI (neutralized to pH 7.0 with HCl) were first separately diluted in 13 µl of 150 mM NaCl. PEI-DNA particles were thereafter obtained by gently mixing the two solutions. Cells, plated in 3.5 cm Petri dishes, were incubated for 1-2 hr in the PEI-DNA solution previously diluted to 0.8 ml with serum-free culture medium. In each culture, part of the cells were exposed to fluorescein-conjugated antisense ONs and part of the cells to fluorescein-conjugated scrambled oligonucleotides (160 nM). ONs were fully phosphorothioated and were designed and produced by Biognostik GmbH (Gottingen, Germany). Specifically, we used antisense ONs against nucleotides 81-98 of the _1E subunit (Soong et al., 1993), corresponding to the N-terminal cytoplasmic region (sequence: GCATATTTCCTGACAATG), against nucleotides 414-431 of the _1A subunit (Starr et al., 1991), corresponding to segment S2 of repeat I (sequence: CCAATGAAATAGGGTTCT), and the corresponding scrambled oligonucleotides (sequences: ACTACTACACTAGACTAC and TCAAAACGAATGCAGTTG, respectively).

Electrophysiology. Whole-cell patch-clamp recordings followed standard techniques (Hamill et al., 1981). Currents were recorded with an Axopatch-200 patch-clamp amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 1 kHz (3 dB; eight-pole Bessel filter), sampled at 5 kHz using a Digidata 1200 interface and pClamp6 software (Axon Instruments), and stored for later analysis on a computer. Compensation (typically 70-80%) for series resistance was generally used, and only data from cells with a voltage error of <3 mV were analyzed. Experiments were performed at room temperature (21-25°C).

To measure the R-type calcium current in isolation, cells were preincubated for 10 min into a recording chamber containing Tyrode's solution supplemented with 1 µM -conotoxin-GVIA (-CgTx-GVIA) (Bachem, Budendorf, Switzerland), 3 µM -conotoxin-MVIIC (-CTx-MVIIC or SNX230 provided by Neurex Corporation, Menlo Park, CA), 5 µM nimodipine (gift from Dr. Hof, Sandoz, Basel, Switzerland), and 0.1 mg/ml cytochrome c (Sigma, St. Louis, MO). After attainment of the whole-cell configuration, cells were perfused with the external recording solution containing: 5 mM BaCl₂, 148 mM TEA-Cl, 10 mM HEPES (adjusted to pH 7.4 with TEA-OH), 5 µM nimodipine, and 0.1 mg/ml cytochrome c. Control experiments in which -CgTx-GVIA and -CTx-MVIIC were sequentially applied at increasing times after perfusion with external solution, established that slow unblocking of the toxins could account for at most 10% of the R-type current measured after 30 min (the maximal duration of our recordings). Internal solution contained (in mM): 100 Cs-methanesulfonate, 5 MgCl₂, 30 HEPES, 10 EGTA, 4 ATP, 0.5 GTP, and 1 mM cAMP (adjusted to pH 7.4 with CsOH). The perfusion system consisted of six microcapillary Teflon tubes glued together and placed inside a standard plastic pipette (Gilson Medical Electronics, Villiers-le-Bel, France) at ~12 mm from the tip (~1.2 mm diameter), which was cut to have a flute beak shape, and positioned close to the cell. The tubes were fed by gravity from reservoirs containing external solution with or without toxins. Switching between different solutions was controlled by solenoid valves. Delay time for complete solution change was <8 sec. Cytocrome c (0.1 mg/ml) was included in all recording solutions to block nonspecific peptide binding sites. Liquid junction potential at the pipette tip was 8 mV (pipette negative), and that between the Tyrode's solution in the experimental chamber and the external recording solution (flowing from the capillary tube) was 4 mV; these two junction potentials should be added to all voltages to obtain the correct values of membrane potential in whole-cell recordings (Neher, 1992). Isolated cells were chosen for recording. The experiment was discarded if cells showed signs of inadequate space clamping, such as notch-like current discontinuities, slow components in the decay of capacitative currents (in response to hyperpolarizing pulses), or slow tails not fully inhibited by nimodipine (Forti and Pietrobon, 1993). Barium currents were corrected on-line for leak and capacitative currents with the P/4 pulse protocol. Averages are given as mean ± SEM. The statistical significance of paired values was tested by an ANOVA, followed by a post hoc t test.

The neurotoxin SNX482, a 41 amino acid peptide present in the venom of the African tarantula Histerocrates gigas (Newcomb et al., 1998) was kindly provided by G. Miljanich and L. Nadasdi (Elan Pharmaceuticals Inc., Menlo Park, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The peptide neurotoxin SNX482 selectively inhibits recombinant calcium channels containing _1E subunits with an IC₅₀ for block of ~30 nM (Newcomb et al., 1998). We assessed the sensitivity to SNX482 of the R-type calcium current of rat cerebellar granule cells in primary culture. To isolate the R-type calcium current, L-, N-, and P/Q-type calcium channels were completely inhibited using saturating concentrations of nimodipine, -CgTx-GVIA, and -Ctx-MVIIC (Hillyard et al., 1992; Zhang et al., 1993; McDonough et al., 1996; Tottene et al., 1996). As shown in Figure 1A (top panel), 10 nM SNX482 slowly inhibited a fraction of the R-type current. No significant further inhibition was observed with 30 nM toxin, but 100 nM toxin inhibited more, indicating the presence of at least two components of R-type current with different sensitivity to SNX482. The lack of significant further inhibition by 200 nM toxin with respect to 100 nM, shown in the bottom panel of Figure 1A, indicates that the R-type calcium current of cerebellar granule cells actually comprises three components with different sensitivity to the toxin. The three R-type current components, R_a, R_b, and R_c, can be seen in the dose-response curve in Figure 1B. The values of IC₅₀ for inhibition of the three components, obtained by fitting the data points with the sum of three Hill equations, were 6, 81, and 654 nM (fractional contributions: 32, 17, and 51%, for R_a, R_b, and R_c, respectively). Figure 1 then shows that the R-type calcium current of rat cerebellar granule cells comprises two components, R_a and R_b, inhibited by concentrations of SNX482 close to those that inhibit recombinant _1E channels, and in addition, a third component, R_c, that can be considered as toxin-resistant because SNX482 is not a selective blocker of _1E channels at the concentrations required to inhibit R_c (Newcomb et al., 1998). The one order of magnitude difference in IC₅₀ for inhibition of components R_a and R_b is reminiscent of the difference in affinity for -agatoxin IVA (-AgaIVA) of P- and Q-type calcium currents (Randall and Tsien, 1995). The two components R_a and R_b differ also in the time course of inhibition by SNX482. As shown by the representative experiment in Figure 4A, the kinetics of inhibition by 30 nM toxin (component R_a) were faster ( of 50 ± 5 sec; n = 10) than those of inhibition by 200 nM toxin, sequentially added after 30 nM (component R_b:  of 74 ± 8 sec; n = 11).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Three components of R-type current with different sensitivity to SNX482 in rat cerebellar granule cells: Ra, Rb, and Rc. Whole-cell recordings of R-type current with 5 mM Ba2+ as charge carrier after inhibition of L-, N-, and P/Q-type currents with saturating concentrations of nimodipine, -CgTx-GVIA, and -CTx-MVIIC (see Materials and Methods). Test depolarizations to 10 mV were delivered every 10 sec from holding potential Vh of 90 mV. A, Plots of peak R-type Ba2+ current, IR, versus time for two representative experiments in which either 5, 10, 30, and 100 nM SNX482 (top panel; cell U89D) or 100, 200, 300, and 500 nM SNX482 (bottom panel; cell U126A) were sequentially applied. Representative average current traces (n = 4) taken at times indicated by a, b, c, d, and e are shown in the insets. Calibration: 25 pA, 20 msec. B, Dose-response curve for SNX482 inhibition of the R-type current, obtained by averaging the fractional inhibitions produced by successive applications of at least three concentrations of toxin in 21 cells. The number of cells from which the average inhibition at any given concentration was obtained is indicated above the symbols. The continuous line is the sum of three Hill equations with na = nb = nc = 2 and Ka of 36 nM (IC50a of 6 nM), Kb of 6.6 µM (IC50b of 81 nM), and Kc of 428 µM (IC50c of 654 nM). A Hill coefficient larger than one was necessary to adequately fit the data points (the fit further improved with n = 3). Failure to completely reach steady-state at low toxin concentrations cannot completely account for the requirement of Hill coefficients greater than one, because, from fitting the time course of inhibition with a single exponential, a maximal 25% underestimation of the inhibition at the lowest toxin concentration can be calculated. A Hill coefficient larger than one (n = 1.4) was also necessary to best fit the dose-response curve for SNX482 inhibition of recombinant calcium channels containing human 1E-d subunits (our unpublished observations).

Recombinant calcium channels containing _1E subunits are characterized by a high sensitivity to Ni²⁺ block (Soong et al., 1993; Schneider et al., 1994; Williams et al., 1994; Zamponi et al., 1996) and by a larger macroscopic current with Ca²⁺ as charge carrier compared with Ba²⁺ (Bourinet et al., 1996; and our unpublished observations with both human _1E-d and _1E-3 subunits). Both observations point to distinct permeation properties of _1E with respect to _1A, _1B, and _1C channels. Tottene et al. (1996) have shown previously that only 50% of the R-type current of cerebellar granule cells shows a high sensitivity to Ni²⁺ block, requiring concentrations of 30-50 µM Ni²⁺ to be fully inhibited. The less sensitive component was inhibited with an IC₅₀ of 153 µM. Figure 2 shows that the R-type current remaining in the presence of 30 µM Ni²⁺ was not further inhibited by concentrations of SNX482 (30 and 100 nM) that completely blocked components R_a and R_b; however, it was partially inhibited by 500 nM SNX482, a concentration that partially inhibited component R_c. These occlusion experiments show that the two R-type current components with relatively high affinity for SNX482 are both very sensitive to Ni²⁺ block, whereas the R-type calcium channels resistant to the toxin have a relatively low sensitivity to Ni²⁺ block.

View larger version (21K): [in this window] [in a new window]   Figure 2.   The Rc current is less sensitive to Ni2+ block than Ra and Rb currents. Whole-cell recordings of R-type current as in Figure 1. Plots of peak R-type Ba2+ current versus time for two representative experiments in which 30 µM Ni2+ was applied, and then either 30 and 100 nM SNX482 were sequentially applied (top panel; cell U213C) or 500 nM SNX482 was applied (bottom panel; cell U137A) in the continuous presence of Ni2+. Representative current traces taken at times indicated by a, b, c, and d are shown in the insets. Calibration: 50 pA, 20 msec. The lack of inhibition by 30 and 100 nM SNX482 means that both components Ra and Rb are fully inhibited by 30 µM Ni2+. The partial inhibition by 500 nM SNX482 means that component Rc is not inhibited by 30 µM Ni2+.

Figure 3 further shows that R_c, the R-type current component resistant to SNX482, has different permeation properties than the two SNX482-sensitive components. Whereas the total R-type current at the peak of the current-voltage (I-V) relationship had similar amplitude with Ba²⁺ or Ca²⁺ as charge carrier (Fig. 3A), the R_c current remaining in the presence of 200 nM SNX482 was larger with Ba²⁺ (Fig. 3B). On average, the ratio I_Ca2+/I_Ba2+ was 0.99 ± 0.02 (n = 4) and 0.72 ± 0.09 (n = 3) for total R-type and R_c currents, respectively (significantly different at p < 0.02). One can then argue that the two SNX482-sensitive current components (R_a and R_b) are larger with Ca²⁺ than with Ba²⁺ as charge carrier and therefore have permeation properties similar to those of recombinant _1E channels (compare also their high sensitivity to Ni²⁺ block). On the other hand, the R_c component has permeation properties similar to those of _1A, _1B, and _1C channels.

View larger version (25K): [in this window] [in a new window]   Figure 3.   The total R-type current has similar amplitude with Ca2+ and Ba2+ as charge carrier, whereas the Rc current is larger with Ba2+. Whole-cell recordings with either 5 mM Ba2+ or 5 mM Ca2+ as charge carrier. Perfusion of the cells with the Ba2+ solution was followed by perfusion with the Ca2+ solution and then again with the Ba2+ solution. Increasing test depolarizations from 50 to + 40 mV were delivered every 5 sec from Vh of 90 mV in each solution. A, Current-voltage relationships of R-type current, IR, and representative traces at increasing depolarizations with either 5 mM Ca2+ (; V of 50 to 0 mV) or 5 mM Ba2+ (; perfused after Ca2+; V of 50 to 10 mV). Calibration: 50 pA, 20 msec. Cell U81A. B, Current-voltage relationships of R-type current in the presence of 200 nM SNX482, IRc, and representative traces at increasing depolarizations with either 5 mM Ca2+ (; V of 50 to 0 mV) or 5 mM Ba2+ (; perfused after Ca2+; V of 50 to 10 mV). Calibration: 50 pA, 20 msec. Cell U214D.

Tottene et al. (1996) have shown previously that at least two different calcium channels, G2 and G3, contribute to the R-type calcium current of cerebellar granule cells. The two channels differ mainly in single channel current and conductance and in voltage range for activation (Forti et al., 1994). G2 channels, with unitary conductance of 15 pS, activate at ~15 mV more negative voltages than G3 channels with unitary conductance of 20 pS. It appears reasonable to predict that G2 channels with unitary conductance similar to that of recombinant _1E channels should support the two SNX-sensitive current components R_a and R_b (with permeation properties similar to those of recombinant _1E channels) and that G3 channels with different unitary conductance should support the SNX-resistant R_c component (with permeation properties different from those of recombinant _1E channels). If this hypothesis is correct, component R_c should activate at more positive voltages than components R_a and R_b. To verify this hypothesis and eventually obtain information on the relative sensitivity to SNX482 of G2 and G3 channels, we performed whole-cell recordings in which 30 and 200 nM SNX482 were sequentially added, and the I-V relationship was measured before and after each toxin addition. According to the dose-response curve in Figure 1, 30 nM toxin completely inhibits component R_a, which is then given by the difference between the R-type current measured in the presence and absence of 30 nM toxin; 200 nM toxin should completely inhibit component R_b, which can then be obtained as the difference between the R-type current measured in the presence of 200 and 30 nM toxin. Component R_c is given by the current remaining in the presence of 200 nM toxin.

As shown by the representative experiment in Figure 4A, the I-V curve in the presence of 30 nM toxin was shifted toward more positive voltages with respect to that in its absence, and the I-V curve in the presence of 200 nM toxin was shifted in the same direction with respect to that in the presence of 30 nM toxin. Figure 4B shows the average normalized I-V curves for the three components; component R_a activates at slightly more negative voltages than R_b, and both R_a and R_b activate at more negative voltages than R_c. The three R-type components had slightly different kinetics of inactivation (5 ± 2, 24 ± 3, and 17 ± 2% decay in 136 msec for R_a, R_b, and R_c, respectively; n = 10) (see Fig. 4A). The different current-voltage relationships, the different kinetics of inactivation, and the different time course of inhibition together support the notion that different calcium channels underlie the R-type current components identified on the basis of the dose-response curve for SNX482. The data are consistent with the conclusion that the channels with the highest affinity for SNX482 correspond to G2, the R channel subtypes activating at more negative voltages. Given the large difference in half-voltage for activation (18 mV) between G2 and G3, the R-type channels resistant to SNX482 most likely correspond to G3, the R channel subtypes with unitary conductance different from that reported for _1E channels. Figure 4C shows that the steady-state inactivation curve of the current remaining in the presence of 200 nM SNX482 was almost identical to that of the total R-type current. Steady-state inactivation of the calcium channels that underlie the three R-type current components then occurred at quite negative voltages (V_1/2 of 76 mV) in a similar voltage range. Steady-state inactivation of both G2 and G3 channels occurred in a similar negative voltage range.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Different voltage-dependence of activation and similar voltage-dependence of inactivation of the three R-type current components. Whole-cell recordings of R-type current with 5 mM Ba2+ as charge carrier. A, Left panel, Plot of peak R-type Ba2+ current, IR, versus time for an experiment in which 30 and 200 nM SNX482 were sequentially applied. Test depolarizations to 10 mV were delivered every 10 sec from Vh of 90 mV. Representative average current traces (n = 4) taken at times indicated by 1, 2, and 3, together with difference trace 1-2, corresponding to the Ra current component, and difference trace 2-3, corresponding to the Rb current component, are shown in the middle inset. Calibration: 50 pA, 20 msec. Right panel, I-V relationships measured at times indicated by symbols in the absence () and presence of 30 nM () and 200 nM () SNX482. Representative traces at increasing test depolarizations (Vt of 50 to 10 mV) are shown in the right inset. Calibration: 50 pA, 20 msec. Cell U198E. B, Average peak normalized current, In, as a function of test voltage, Vt, for the three components Ra (; n = 12), Rb (; n = 13), and Rc (; n = 13). For each cell, the current was normalized with respect to maximal peak current. At each voltage, Ra is obtained as difference between the R-type currents measured in the presence and absence of 30 nM toxin; Rb is obtained as difference between the R-type currents measured in the presence of 200 and 30 nM toxin, and Rc is the R-type current remaining in the presence of 200 nM toxin. C, Average peak normalized R-type current, In, at 10 mV as a function of holding potential, Vh, in the absence (total R, ; n = 8) and presence of 200 nM SNX482 (Rc, ; n = 6). For each cell, the current was normalized with respect to the peak current at Vh of 110 mV. The data points were best fit by a Boltzmann distribution function of the form In = In max × (1 + exp ((V  V1/2)/k))1 with V1/2 of 76 mV and k of 11 mV for both total R and Rc currents.

Both the pharmacological and permeation properties of the R-type channels resistant to SNX482 of rat cerebellar granule cells are quite different from those of recombinant channels containing _1E subunits. _1B, _1A, or _1E subunits with low affinity for their specific toxins or an unknown ₁ subunit appear equally likely as the pore-forming subunits of these channels. To investigate the molecular basis of the SNX482-resistant R-type channels and to directly show that _1E subunits are the pore-forming subunits of the SNX482-sensitive R-type channels, we turned to an antisense strategy.

Cerebellar granule cells were transfected with fluorescein-conjugated anti-_1E antisense ON 1 d after plating. R-type calcium current densities were measured at 3, 4, and 5 d after transfection. At each day, control R-type current densities were measured in cells from the same culture transfected with fluorescein-conjugated scrambled ON. Transfected neurons were identified by their fluorescent nuclei. As shown in Figure 5A, _1E subunit knock-down strongly decreased the R-type current of cerebellar granule cells. A maximum decrease of 87% was achieved 4 d after transfection (from 23 ± 4 to 3 ± 1 pA/pF; p 0.001). After 3 d, the current was already 75% reduced (p < 0.001), suggesting a turnover time of ~3 d for these ₁ subunits. Figure 5B shows that _1E subunit knock-down decreased to a similar extent both the fraction of R-type current inhibited by 30 µM Ni²⁺ (corresponding to components R_a and R_b) and the fraction of R-type current remaining not inhibited (corresponding to component R_c). Analysis of the development of the different components of control R-type current with increasing days in culture shows that the small increase in total R-type current observed between days 3 and 4 after transfection (corresponding to the fourth and fifth day in culture) is caused by an increased expression of the SNX482-resistant R-type channels with low affinity for Ni²⁺. The R-type current remaining in the presence of 30 µM Ni²⁺ increased 78% (p < 0.04) between days 3 and 4 after transfection, whereas the R-type current inhibited by the same concentration of Ni²⁺ did not change.

View larger version (17K): [in this window] [in a new window]   Figure 5.   1E subunit knock-down by specific antisense oligonucleotides decreases to a similar extent the three R-type current components of rat cerebellar granule cells. Whole-cell recordings of R-type current as in Figure 1. Data are pooled from four neuronal cultures. In each culture, current densities were measured in both antisense ON- and scrambled ON-transfected cells at 3, 4, and 5 d after transfection. A, Total R-type current densities as a function of time after transfection, measured in cerebellar granule cells transfected with 1E antisense ON (black bars) and with scrambled ON (white bars). Peak R-type current densities were measured 3 min after entering into the whole-cell configuration. Antisense ON: n = 13, 15, and 8 at days 3, 4, and 5, respectively. Scrambled ON: n = 12, 11, and 11 at days 3, 4, and 5, respectively. The reduction of the R-type current in antisense-transfected cells was significant at each day in culture (75%, p < 0.001; 87%, p 0.001; and 53%, p < 0.02 at day 3, 4, and 5, respectively). B, R-type current densities as a function of time after transfection, measured in the presence of 30 µM Ni2+ (corresponding to component Rc; left panel) and inhibited by 30 µM Ni2+ (corresponding to components Ra and Rb; right panel) in cerebellar granule cells transfected with 1E antisense ON (black bars) and with scrambled ON (white bars). Ni2+ was applied 3 min after entering into the whole-cell configuration. Antisense ON: n = 11, 11, and 5 at days 3, 4, and 5, respectively. Scrambled ON: n = 10, 6, and 8 at days 3, 4, and 5, respectively. The reduction of the different R-type current components in the antisense-transfected neurons was significant at each day in culture (Rc: 74%, p < 0.001; 86%, p 0.001; 69%, p < 0.02; Ra and Rb: 72%, p < 0.02; 80%, p < 0.001; 64%, p < 0.02 at 3, 4, and 5 d, respectively).

The specificity of the anti-_1E antisense ON was tested by investigating its effect on the N-type calcium current. Figure 6A shows that the current density inhibited by 1 µM -CgTx-GVIA was similar in cells transfected with antisense and scrambled ON, whereas in the same cells the R-type current was 73% decreased in antisense-transfected cells. As an additional test, we ascertained that an anti-_1A antisense oligonucleotide, which proved to be effective in decreasing the P-type current in Purkinje cells (Gillard et al., 1997), did not affect the R-type current of cerebellar granule cells. Figure 6B shows that the R-type current was similar in cells transfected with anti-_1A antisense and scrambled ON.

View larger version (16K): [in this window] [in a new window]   Figure 6.   The N-type current is not affected by 1E antisense ONs, and the R-type current is not affected by 1A antisense ONs. Whole-cell recordings with 5 mM Ba2+ as charge carrier. Voltage protocol as in Figure 1. A, (N + R)-type Ba2+ current densities were measured 4 d after transfection in cerebellar granule cells transfected with 1E antisense ON (black bars) and with scrambled ON (white bars) in the continuous presence of 5 µM nimodipine after incubation of the neurons with 3 µM -CTx-MVIIC to irreversibly block P/Q-type channels; -CgTx-GVIA (1 µM) was then applied. N-type and R-type current densities were obtained from the current densities inhibited by -CgTx-GVIA and remaining in the presence of -CgTx-GVIA, respectively. N-type current densities were similar in antisense ON- (n = 7) and scrambled ON- (n = 8) transfected cells, whereas R-type currents were 73% smaller in antisense ON-transfected cells (p < 0.002). B, R-type current densities measured 4-5 d after transfection in cerebellar granule cells transfected with 1A antisense ON (black bars; n = 10) and with scrambled ON (white bars; n = 9) were similar.

Thus, quite unexpectedly, the antisense data show that _1E subunits form the pore of the R subtypes of calcium channels of rat cerebellar granule cells with pharmacological and permeation properties different from those of recombinant channels containing _1E subunits. Moreover, they directly show that the two SNX482-sensitive R subtypes both contain _1E subunits as pore-forming subunits.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Using an antisense strategy combined with electrophysiology and the new selective pharmacological tool provided by SNX482, we show that rat cerebellar granule cells coexpress calcium channels containing _1E subunits with widely different pharmacological and biophysical (including permeation) properties.

Previous work on recombinant calcium channels containing _1E subunits has led to the identification of a few specific properties distinguishing them from high-voltage activated _1A, _1B, and _1C channels. The specific properties, common to all the different isoforms of _1E studied so far, include the following (1) a high sensitivity to Ni²⁺ block (Soong et al., 1993; Schneider et al., 1994; Williams et al., 1994); (2) the peptide neurotoxin SNX482 as the only selective, high-affinity antagonist (Newcomb et al., 1998, and our unpublished observations), (3) a larger macroscopic current with Ca²⁺ than with Ba²⁺ as charge carrier (Bourinet et al., 1996; and our unpublished observations with both human _1E-d and _1E-3 subunits), and (4) a single channel conductance of 12-15 pS (Schneider et al., 1994; Wakamori et al., 1994; Bourinet et al., 1996).

Here, we show that these properties are shared by the native calcium channels containing _1E subunits, which account for the two SNX482-sensitive components of the R-type current of rat cerebellar granule cells. The one order of magnitude difference in IC₅₀ for inhibition of these two components (6 and 81 nM) is reminiscent of the difference in affinity for -AgaIVA of P- and Q-type calcium currents (Randall and Tsien, 1995). It has been shown that _1A subunits are the pore-forming subunits of both P- and Q-type channels (Gillard et al., 1997; Piedras-Renteria and Tsien, 1998) and that alternative splicing of the _1A gene may give rise to calcium channels with pharmacological and biophysical properties similar to those of P- and Q-type channels (Bourinet et al., 1999). Likewise, alternative splicing of the _1E gene may possibly account for the different sensitivity to SNX482 of the two R-type current components. Alternatively, the different pharmacology may be attributable to different combinations with auxiliary subunits (Moreno et al., 1997).

Single channel recordings have shown previously that two calcium channels, G2 and G3, with different unitary conductance and voltage-dependence of activation, contribute to the R-type calcium current of rat cerebellar granule cells (Forti et al., 1994; Tottene et al., 1996). G2 channels have unitary conductance and current (15 pS, 0.6 pA at 0 mV) similar to those of recombinant _1E channels, whereas G3 channels have larger unitary conductance and current (20 pS, 0.8 pA at 0 mV). G2 channels activate at ~15 mV more negative voltages than G3. A comparable difference in voltage range of activation has been found here between the component of R-type current most sensitive to SNX482 and that resistant to SNX482, the first activating at more negative voltages than the latter. Our data are consistent with the conclusion that the R-type channels with high affinity for SNX482 correspond to G2, and those with very low affinity for SNX482 correspond to G3. Given the small difference in voltage-dependence of activation of the two SNX482-sensitive components of R-type current, it appears likely that, at the single channel level, the channels underlying these two components were lumped together under the name of G2. This conclusion is further supported by the fact that, in cell-attached patches, G2 channels were observed more frequently than G3 channels (Forti et al., 1994), and R-type channels different from G2 and G3 were extremely rare (our unpublished observations).

The component of the R-type calcium current of rat cerebellar granule cells resistant to SNX482 shows none of the "specific" properties of recombinant _1E channels; it is not very sensitive to Ni²⁺ block, it is larger with Ba²⁺ than with Ca²⁺ as charge carrier, and, most likely, it is supported by G3 channels with conductance of 20 pS. Nonetheless, strikingly, this component is suppressed after transfection of the neurons with a specific anti-_1E antisense oligonucleotide. Therefore, in addition to calcium channels containing _1E subunits with permeation properties considered as typical of _1E subunits, rat cerebellar granule cells express calcium channels containing _1E subunits with permeation properties more typical of _1A, _1B, or _1C subunits. It is highly unlikely that these permeation properties are caused by a particular combination with auxiliary subunits, because  and ₂- subunits do not appear to affect single channel conductance and permeation of recombinant ₁ channels (Wakamori et al., 1993, 1999; and our unpublished observations). Most likely, these peculiar native R-type channels contain a novel _1E variant, whose properties have not been studied in heterologous expression systems.

The component of R-type current resistant to SNX482, accounting for ~50% of the R-type current of our cerebellar granule cells, has many properties in common with the original R-type current described in the same neurons by Zhang et al. (1993). The common properties include the ratio I_Ca/I_Ba lower than 1, the relatively low sensitivity to Ni²⁺ block, the resistance to SNX482 (Newcomb et al., 1998), and the reduction by specific anti-_1E antisense oligonucleotides (Piedras-Renteria and Tsien, 1998). However, in cerebellar granule cells cultured under the conditions of Zhang et al. (1993), there was no evidence for the presence of additional components of R-type current inhibited with high affinity by SNX482 (Newcomb et al., 1998). The most likely explanation is the expression of different _1E splice variants in neurons cultured under different conditions and obtained from rats of different age. Similarly, expression of different _1A splice variants (and/or different  subunits) may explain the P-type pharmacology found by Tottene et al. (1996) in contrast with the Q-type pharmacology found by Randall and Tsien (1995) in the same neurons (Moreno et al., 1997; Bourinet et al., 1999).

Reverse transcription-PCR analysis of the mRNA isolated from our primary cultures of cerebellar granule cells has shown the expression of up to six different _1E isoforms alternatively spliced in the II-III loop and the C terminus, some of which had not been cloned before (Schramm et al., 1999). There is evidence for different relative expression of these isoforms in different brain regions and during development (Pereverzev et al., 1998; Schramm et al., 1999). Two of the isoforms do not show differences in biophysical properties (Pereverzev et al., 1998). The functional properties of the other four isoforms remain unknown. Splice variants of _1A, _1B, and _1C with different kinetics and/or voltage-dependence of the macroscopic calcium current have been described previously (Lin et al., 1997; Soldatov et al., 1997; Bourinet et al., 1999; Hans et al., 1999). However, splice variants of calcium channel ₁ subunits with different permeation properties and different unitary conductance have never before been reported.

Labeling with antibodies of brain slices has shown localization of _1E subunits in both cell bodies and dendrites of many types of neurons (Volsen et al., 1995; Yokoyama et al., 1995) and more recently also in calyx-type synaptic terminals of the brainstem (Wu et al., 1999). Wu et al. (1998) have shown that R-type channels contribute to action potential-evoked transmitter release at these synapses. Most likely, R-type channels participate in controlling evoked release in many other central synapses (Turner et al., 1993), including cerebellar parallel fiber synapses (Mintz et al., 1995). The localization in cell bodies and dendrites suggest additional postsynaptic roles of R-type channels, e.g., G2-type channels with a relatively low threshold of activation might have a role in synaptic integration and the generation of calcium spikes (D'Angelo et al., 1997). It is reasonable to hypothesize that R-type channels with different voltage-dependence of activation and different unitary conductance as those coexpressed in cerebellar granule cells may have a specialized function in different neuronal processes. Alternative splicing of _1E subunits may allow different subcellular localizations, different modulation, and different binding to specific membrane proteins of the different R-type channels. Indeed, in the case of _1A subunits, it has been shown that the II-III loop and the C-terminal region, the two regions alternatively spliced in the _1E isoforms expressed in cerebellar granule cells, are involved in binding important proteins, such as syntaxin (Rettig et al., 1996) and calmodulin (Lee et al., 1999), respectively, and that alternatively spliced II-III loop isoforms have a different ability to bind syntaxin (Rettig et al., 1996) and have a different subcellular distribution (Sakurai et al., 1996).

	FOOTNOTES

Received Aug. 9, 1999; revised Oct. 18, 1999; accepted Oct. 20, 1999.

This work was supported by Telethon-Italy Grant 720 to D.P. A.T. was supported by the Lilly Postdoctoral Research Program. We thank Drs. G. Miljanich and L. Nadasdi of Elan Pharmaceuticals Inc. for providing SNX483, and Carlo Berizzi and Francesca Bolcato for help in performing some of the experiments.

Correspondence should be addressed to Daniela Pietrobon, Department of Biomedical Sciences, University of Padova, V.le G. Colombo 3, 35121 Padova, Italy. E-mail: dani{at}civ.bio.unipd.it.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Bourinet E, Zamponi GW, Stea A, Soong TW, Lewis BA, Jones LP, T YD, Snutch TP (1996) The _1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels. J Neurosci 16:4983-4993[Abstract/Free Full Text].
• Bourinet E, Soong TW, Sutton K, Slaymaker S, Mathews E, Monteil A, Zamponi GW, Nargeot J, Snutch TP (1999) Splicing of _1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nature Neuroscience 2:407-415[Web of Science][Medline].
• D'Angelo E, De Filippi G, Rossi P, Taglietti V (1997) Synaptic activation of Ca²⁺ action potentials in immature rat cerebellar granule cells in situ. J Neurophysiol 78:1631-1642[Abstract/Free Full Text].
• Dunlap K, Luebke JI, Turner TJ (1995) Exocytotic Ca²⁺ channels in mammalian central neurons. Trends Neurosci 18:89-98[Web of Science][Medline].
• Forti L, Pietrobon D (1993) Functional diversity of L-type calcium channels in rat cerebellar neurons. Neuron 10:437-450[Web of Science][Medline].
• Forti L, Tottene A, Moretti A, Pietrobon D (1994) Three novel types of voltage-dependent calcium channels in rat cerebellar neurons. J Neurosci 14:5243-5256[Abstract].
• Gillard SE, Volsen SG, Smith W, Beattie RE, Bleakman D, Lodge D (1997) Identification of pore-forming subunit of P-type calcium channels: an antisense study on rat cerebellar Purkinje cells in culture. Neuropharmacology 36:405-409[Web of Science][Medline].
• Hamill O, Marty A, Neher E, Sakmann B, Sigworth F (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85-100[Web of Science][Medline].
• Hans M, Urrutia A, Deal C, Brust PF, Stauderman K, Ellis SB, Harpold MM, Johnston EC, Williams ME (1999) Structural elements in domain IV that influence biophysical and pharmacological properties of human _1A-containing high-voltage-activated calcium channels. Biophys J 76:1384-1400[Web of Science][Medline].
• Hilaire C, Diochot S, Desmadryl G, Richard S, Valmier J (1997) Toxin-resistant calcium currents in embryonic mouse sensory neurons. Neuroscience 80:267-276[Web of Science][Medline].
• Hillyard DR, Monje VD, Mintz IM, Bean BP, Nadasdi L, Ramachandran J, Miljanich G, Azimi-Zoonooz A, McIntosh JM, Cruz LJ, Imperial JS, Oliveira BM (1992) A new Conus peptide ligand for mammalian presynaptic Ca²⁺ channels. Neuron 9:69-77[Web of Science][Medline].
• Lambert RC, Maulet Y, Dupont JL, Mykita S, Craig P, Volsen S, Feltz A (1996) Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture: probing Ca²⁺ channel structure and function with antisense oligonucleotides. Mol Cell Neurosci 7:239-246[Web of Science][Medline].
• Lee A, Wong ST, Gallagher D, Li B, Storm DR, Scheuer T, Catterall WA (1999) Ca²⁺/calmodulin binds to and modulates P/Q-type calcium channels. Nature 399:155-159[Medline].
• Levi G, Aloisi M, Ciotti M, Gallo V (1984) Autoradiographic localization and depolarization-induced release of amino acids in differentiating granule cells cultures. Brain Res 290:77-86[Web of Science][Medline].
• Lin Z, Haus S, Edgerton J, Lipscombe D (1997) Identification of functionally distinct isoforms of the N-type Ca²⁺ channel in rat sympathetic ganglia and brain. Neuron 18:153-166[Web of Science][Medline].
• Magnelli V, Baldelli P, Carbone E (1998) Antagonists-resistant calcium currents in rat embryo motoneurons. Eur J Neurosci 10:1810-1825[Web of Science][Medline].
• McDonough SI, Swartz KJ, Mintz IM, Boland LM, Bean BP (1996) Inhibition of calcium channels in rat central and peripheral neurons by -conotoxin MVIIC. J Neurosci 16:2612-2623[Abstract/Free Full Text].
• Mintz IM, Sabatini BL, Regehr WG (1995) Calcium control of transmitter release at a cerebellar synapse. Neuron 15:675-688[Web of Science][Medline].
• Moreno H, Rudy B, Llinas R (1997) subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line. Proc Natl Acad Sci USA 94:14042-14047[Abstract/Free Full Text].
• Neher E (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207:123-131[Web of Science][Medline].
• Newcomb R, Szoke B, Palma A, Wang G, Chen X, Hopkins W, Cong R, Miller J, Urge L, Tarczy-Hornoch K, Loo JA, Dooley DJ, Nadasdi L, Tsien RW, Lemos J, Miljanich G (1998) Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 37:15353-15362[Medline].
• Nooney JM, Lambert RC, Feltz A (1997) Identifying neuronal non-L Ca²⁺ channelsmore than stamp collecting? Trends Pharmacol Sci 18:363-371[Medline].
• Pereverzev A, Klockner U, Henry M, Grabsch H, Vajna R, Olyschlager S, Viatchenko-Karpinski S, Schroder R, Hescheler J, Schneider T (1998) Structural diversity of the voltage-dependent Ca²⁺ channel _1E-subunit. Eur J Neurosci 10:916-925[Web of Science][Medline].
• Piedras-Renteria ES, Tsien RW (1998) Antisense oligonucleotides against _1E reduce R-type calcium currents in cerebellar granule cells. Proc Natl Acad Sci USA 95:7760-7765[Abstract/Free Full Text].
• Randall A, Tsien RW (1995) Pharmacological dissection of multiple types of Ca²⁺ channel currents in rat cerebellar granule neurons. J Neurosci 15:2995-3012[Abstract].
• Rettig J, Sheng ZH, Kim DK, Hodson CD, Snutch TP, Catterall WA (1996) Isoform-specific interaction of the _1A subunits of brain Ca²⁺ channels with the presynaptic proteins syntaxin and SNAP-25. Proc Natl Acad Sci USA 93:7363-7368[Abstract/Free Full Text].
• Sakurai T, Westenbroek RE, Rettig J, Hell J, Catterall WA (1996) Biochemical properties and subcellular distribution of the BI and rbA isoforms of _1A subunits of brain calcium channels. J Cell Biol 134:511-528[Abstract/Free Full Text].
• Schneider T, Wei X, Olcese R, Costantin JL, Neely A, Palade P, Perez-Reyes E, Qin N, Zhou J, Crawford GD, Smith RG, Appel SH, Stefani E, Birnbaumer L (1994) Molecular analysis and functional expression of the human type E neuronal Ca²⁺ channel ₁ subunit. Receptors Channels 2:255-270[Web of Science][Medline].
• Schramm M, Vajna R, Preverzev A, Tottene A, Klockner U, Pietrobon D, Hescheler J, Schneider T (1999) Isoforms of _1E voltage-gated calcium channels in rat cerebellar granule cells: detection of major calcium channel ₁ transcripts by reverse transcription-polymerase chain reaction. Neuroscience 92:565-575[Web of Science][Medline].
• Soldatov NM, Zuhlke RD, Bouron A, Reuter H (1997) Molecular structures involved in L-type calcium channel inactivation. J Biol Chem 272:3560-3566[Abstract/Free Full Text].
• Soong TW, Stea A, Hodson CD, Dubel SF, Vincent SR, Snutch TP (1993) Structure and functional expression of a member of the low voltage-activated calcium channel family. Science 260:1133-1136[Abstract/Free Full Text].
• Starr TVB, Prystay W, Snutch TP (1991) Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc Natl Acad Sci USA 88:5621-5625[Abstract/Free Full Text].
• Tottene A, Moretti A, Pietrobon D (1996) Functional diversity of P-type and R-type calcium channels in rat cerebellar neurons. J Neurosci 16:6353-6363[Abstract/Free Full Text].
• Turner TJ, Adams ME, Dunlap K (1993) Multiple Ca²⁺ channel types coexist to regulate synaptosomal neurotransmitter release. Proc Natl Acad Sci USA 90:9518-9522[Abstract/Free Full Text].
• Volsen SG, Day NC, McCormack AL, Smith W, Craig PJ, Beattie R, Ince PG, Shaw PJ, Ellis SB, Gillespie A, Harpold MM, Lodge D (1995) The expression of neuronal voltage-dependent calcium channels in human cerebellum. Mol Brain Res 34:271-282[Medline].
• Wakamori M, Mikala G, Schwartz A, Yatani A (1993) Single-channel analysis of a cloned human heart L-type Ca²⁺ channel ₁ subunit and the effects of a cardiac  subunit. Biochem Biophys Res Commun 196:1170-1176[Web of Science][Medline].
• Wakamori M, Niidome T, Furutama D, Furuichi T, Mikoshiba K, Fujita Y, Tanaka I, Katayama K, Yatani A, Schwartz A, Mori Y (1994) Distinctive functional properties of the neuronal BII (class E) calcium channel. Receptors Channels 2:303-314[Web of Science][Medline].
• Wakamori M, Mikala G, Mori Y (1999) Auxiliary subunits operate as a molecular switch in determining gating behaviour of the unitary N-type Ca²⁺ channel current in Xenopus oocytes. J Physiol (Lond) 517.3:659-672[Abstract/Free Full Text].
• Williams ME, Marubio LM, Deal CR, Hans M, Brust PF, Philipson LH, Miller RJ, Johnson EC, Harpold MM, Ellis SB (1994) Structure and functional characterization of neuronal _1E calcium channel subtypes. J Biol Chem 269:22347-22357[Abstract/Free Full Text].
• Wu LG, Borst JGG, Sakmann B (1998) R-type Ca²⁺ currents evoke transmitter release at a rat central synapse. Proc Natl Acad Sci USA 95:4720-4725[Abstract/Free Full Text].
• Wu LG, Westenbroek RE, Borst JGG, Catterall WA, Sakmann B (1999) Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses. J Neurosci 19:726-736[Abstract/Free Full Text].
• Yokoyama CT, Westenbroek RE, Hell JW, Soong TW, Snutch TP, Catterall WA (1995) Biochemical properties and subcellular distribution of the neuronal class E calcium channel ₁ subunit. J Neurosci 15:6419-6432[Abstract/Free Full Text].
• Zamponi GW, Bourinet E, Snutch TP (1996) Nickel block of a family of neuronal calcium channels: subtype- and subunit-dependent action at multiple sites. J Membrane Biol 151:77-90[Web of Science][Medline].
• Zhang JF, Randall AD, Ellinor PT, Horne WA, Sather WA, Tanabe T, Schwarz TL, Tsien RW (1993) Distinctive pharmacology and kinetics of cloned neuronal Ca²⁺ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32:1075-1088[Web of Science][Medline].

---

Copyright © 2000 Society for Neuroscience  0270-6474/0/201171-08$05.00/0

This article has been cited by other articles:

				X. Xu and H. M. Colecraft Engineering Proteins for Custom Inhibition of CaV Channels Physiology, August 1, 2009; 24(4): 210 - 218. [Abstract] [Full Text] [PDF]

				P. M. Joksovic, M. Weiergraber, W. Lee, H. Struck, T. Schneider, and S. M. Todorovic Isoflurane-Sensitive Presynaptic R-Type Calcium Channels Contribute to Inhibitory Synaptic Transmission in the Rat Thalamus J. Neurosci., February 4, 2009; 29(5): 1434 - 1445. [Abstract] [Full Text] [PDF]

				M. E. Quinlan, C. O. Alberto, and M. Hirasawa Short-term potentiation of mEPSCs requires N-, P/Q- and L-type Ca2+ channels and mitochondria in the supraoptic nucleus J. Physiol., July 1, 2008; 586(13): 3147 - 3161. [Abstract] [Full Text] [PDF]

				K. Alvina and K. Khodakhah Selective regulation of spontaneous activity of neurons of the deep cerebellar nuclei by N-type calcium channels in juvenile rats J. Physiol., May 15, 2008; 586(10): 2523 - 2538. [Abstract] [Full Text] [PDF]

				L. Li, J. Bischofberger, and P. Jonas Differential Gating and Recruitment of P/Q-, N-, and R-Type Ca2+ Channels in Hippocampal Mossy Fiber Boutons J. Neurosci., December 5, 2007; 27(49): 13420 - 13429. [Abstract] [Full Text] [PDF]

				S.-K. Yang, H. C. Parkington, J. Epelbaum, D. J. Keating, and C. Chen Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2 Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1863 - E1870. [Abstract] [Full Text] [PDF]

				M. Weiergraber, M. Henry, K. Radhakrishnan, J. Hescheler, and T. Schneider Hippocampal Seizure Resistance and Reduced Neuronal Excitotoxicity in Mice Lacking the Cav2.3 E/R-Type Voltage-Gated Calcium Channel J Neurophysiol, May 1, 2007; 97(5): 3660 - 3669. [Abstract] [Full Text] [PDF]

				Y. Yaari, C. Yue, and H. Su Recruitment of apical dendritic T-type Ca2+ channels by backpropagating spikes underlies de novo intrinsic bursting in hippocampal epileptogenesis J. Physiol., April 15, 2007; 580(2): 435 - 450. [Abstract] [Full Text] [PDF]

				Z. Fang, C.-K. Park, H. Y. Li, H. Y. Kim, S.-H. Park, S. J. Jung, J. S. Kim, A. Monteil, S. B. Oh, and R. J. Miller Molecular Basis of Cav2.3 Calcium Channels in Rat Nociceptive Neurons J. Biol. Chem., February 16, 2007; 282(7): 4757 - 4764. [Abstract] [Full Text] [PDF]

				E. W. Tringham, C. E. Payne, J. R. B. Dupere, and M. M. Usowicz Maturation of rat cerebellar Purkinje cells reveals an atypical Ca2+ channel current that is inhibited by {omega}-agatoxin IVA and the dihydropyridine (-)-(S)-Bay K8644 J. Physiol., February 1, 2007; 578(3): 693 - 714. [Abstract] [Full Text] [PDF]

				U. Meza, A. Thapliyal, R. A. Bannister, and B. A. Adams Neurokinin 1 Receptors Trigger Overlapping Stimulation and Inhibition of CaV2.3 (R-Type) Calcium Channels Mol. Pharmacol., January 1, 2007; 71(1): 284 - 293. [Abstract] [Full Text] [PDF]

				M. Kato, N. Tanaka, S. Usui, and Y. Sakuma The SK channel blocker apamin inhibits slow afterhyperpolarization currents in rat gonadotropin-releasing hormone neurones J. Physiol., July 15, 2006; 574(2): 431 - 442. [Abstract] [Full Text] [PDF]

				R. A. Anderson, K. A. Feathergill, D. P. Waller, and L. J. D. Zaneveld SAMMA Induces Premature Human Acrosomal Loss by Ca2+ Signaling Dysregulation J Androl, July 1, 2006; 27(4): 568 - 577. [Abstract] [Full Text] [PDF]

				Y.-J. Won, K. Whang, I. D. Kong, K.-S. Park, J.-W. Lee, and S.-W. Jeong Expression Profiles of High Voltage-Activated Calcium Channels in Sympathetic and Parasympathetic Pelvic Ganglion Neurons Innervating the Urogenital System J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1064 - 1071. [Abstract] [Full Text] [PDF]

				P. M Joksovic, D. A Bayliss, and S. M Todorovic Different kinetic properties of two T-type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane J. Physiol., July 1, 2005; 566(1): 125 - 142. [Abstract] [Full Text] [PDF]

				A. E. Metz, T. Jarsky, M. Martina, and N. Spruston R-Type Calcium Channels Contribute to Afterdepolarization and Bursting in Hippocampal CA1 Pyramidal Neurons J. Neurosci., June 15, 2005; 25(24): 5763 - 5773. [Abstract] [Full Text] [PDF]

				R. Felix Molecular physiology and pathology of Ca2+-conducting channels in the plasma membrane of mammalian sperm Reproduction, March 1, 2005; 129(3): 251 - 262. [Abstract] [Full Text] [PDF]

				A. Rocher, E. Geijo-Barrientos, A. I. Caceres, R. Rigual, C. Gonzalez, and L. Almaraz Role of voltage-dependent calcium channels in stimulus-secretion coupling in rabbit carotid body chemoreceptor cells J. Physiol., January 15, 2005; 562(2): 407 - 420. [Abstract] [Full Text] [PDF]

				P. Isope and T. H. Murphy Low threshold calcium currents in rat cerebellar Purkinje cell dendritic spines are mediated by T-type calcium channels J. Physiol., January 1, 2005; 562(1): 257 - 269. [Abstract] [Full Text] [PDF]

				X. Bian, X. Zhou, and J. J. Galligan R-type calcium channels in myenteric neurons of guinea pig small intestine Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G134 - G142. [Abstract] [Full Text] [PDF]

				H. Daniel, A. Rancillac, and F. Crepel Mechanisms underlying cannabinoid inhibition of presynaptic Ca2+ influx at parallel fibre synapses of the rat cerebellum J. Physiol., May 15, 2004; 557(1): 159 - 174. [Abstract] [Full Text] [PDF]

				M. Watanabe, Y. Sakuma, and M. Kato High Expression of the R-Type Voltage-Gated Ca2+ Channel and Its Involvement in Ca2+-Dependent Gonadotropin-Releasing Hormone Release in GT1-7 Cells Endocrinology, May 1, 2004; 145(5): 2375 - 2383. [Abstract] [Full Text] [PDF]

				R. A. Bannister, K. Melliti, and B. A. Adams Differential Modulation of CaV2.3 Ca2+ Channels by G{alpha}q/11-Coupled Muscarinic Receptors Mol. Pharmacol., February 1, 2004; 65(2): 381 - 388. [Abstract] [Full Text] [PDF]

				A. Brandt, J. Striessnig, and T. Moser CaV1.3 Channels Are Essential for Development and Presynaptic Activity of Cochlear Inner Hair Cells J. Neurosci., November 26, 2003; 23(34): 10832 - 10840. [Abstract] [Full Text] [PDF]

				M. Kato, K. Ui-Tei, M. Watanabe, and Y. Sakuma Characterization of Voltage-Gated Calcium Currents in Gonadotropin-Releasing Hormone Neurons Tagged with Green Fluorescent Protein in Rats Endocrinology, November 1, 2003; 144(11): 5118 - 5125. [Abstract] [Full Text] [PDF]

				F. J. Urbano, E. S. Piedras-Renteria, K. Jun, H.-S. Shin, O. D. Uchitel, and R. W. Tsien Altered properties of quantal neurotransmitter release at endplates of mice lacking P/Q-type Ca2+ channels PNAS, March 18, 2003; 100(6): 3491 - 3496. [Abstract] [Full Text] [PDF]

				R. K. Cloues and W. A. Sather Afterhyperpolarization Regulates Firing Rate in Neurons of the Suprachiasmatic Nucleus J. Neurosci., March 1, 2003; 23(5): 1593 - 1604. [Abstract] [Full Text] [PDF]

				M. Cataldi, E. Perez-Reyes, and R. W. Tsien Differences in Apparent Pore Sizes of Low and High Voltage-activated Ca2+ Channels J. Biol. Chem., November 22, 2002; 277(48): 45969 - 45976. [Abstract] [Full Text] [PDF]

				D. Sochivko, A. Pereverzev, N. Smyth, C. Gissel, T. Schneider, and H. Beck The CaV2.3 Ca2+ channel subunit contributes to R-Type Ca2+ currents in murine hippocampal and neocortical neurones J. Physiol., August 1, 2002; 542(3): 699 - 710. [Abstract] [Full Text] [PDF]

				S.-C. Lee, S. Choi, T. Lee, H.-L. Kim, H. Chin, and H.-S. Shin Molecular basis of R-type calcium channels in central amygdala neurons of the mouse PNAS, February 14, 2002; (2002) 52697799. [Abstract] [Full Text] [PDF]

				S. Gasparini, A. M. Kasyanov, D. Pietrobon, L. L. Voronin, and E. Cherubini Presynaptic R-Type Calcium Channels Contribute to Fast Excitatory Synaptic Transmission in the Rat Hippocampus J. Neurosci., November 15, 2001; 21(22): 8715 - 8721. [Abstract] [Full Text] [PDF]

				H. Liang and K. S. Elmslie Ef-Current Contributes to Whole-Cell Calcium Current in Low Calcium in Frog Sympathetic Neurons J Neurophysiol, September 1, 2001; 86(3): 1156 - 1163. [Abstract] [Full Text] [PDF]

				W. Li, C. Thaler, and P. Brehm Calcium Channels in Xenopus Spinal Neurons Differ in Somas and Presynaptic Terminals J Neurophysiol, July 1, 2001; 86(1): 269 - 279. [Abstract] [Full Text] [PDF]

				H.-y. Jung, N. P. Staff, and N. Spruston Action Potential Bursting in Subicular Pyramidal Neurons Is Driven by a Calcium Tail Current J. Neurosci., May 15, 2001; 21(10): 3312 - 3321. [Abstract] [Full Text] [PDF]

				T. R. Neelands, A. P. J. King, and R. L. Macdonald Functional Expression of L-, N-, P/Q-, and R-Type Calcium Channels in the Human NT2-N Cell Line J Neurophysiol, December 1, 2000; 84(6): 2933 - 2944. [Abstract] [Full Text] [PDF]

				S. M. Wilson, P. T. Toth, S. B. Oh, S. E. Gillard, S. Volsen, D. Ren, L. H. Philipson, E. C. Lee, C. F. Fletcher, L. Tessarollo, et al. The Status of Voltage-Dependent Calcium Channels in alpha 1E Knock-Out Mice J. Neurosci., December 1, 2000; 20(23): 8566 - 8571. [Abstract] [Full Text] [PDF]

				V. Leuranguer, A. Monteil, E. Bourinet, G. Dayanithi, and J. Nargeot T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2540 - H2548. [Abstract] [Full Text] [PDF]

				K. Melliti, U. Meza, and B. Adams Muscarinic Stimulation of alpha 1E Ca Channels Is Selectively Blocked by the Effector Antagonist Function of RGS2 and Phospholipase C-beta 1 J. Neurosci., October 1, 2000; 20(19): 7167 - 7173. [Abstract] [Full Text] [PDF]

				L. Forti, C. Pouzat, and I. Llano Action potential-evoked Ca2+ signals and calcium channels in axons of developing rat cerebellar interneurones J. Physiol., August 15, 2000; 527(1): 33 - 48. [Abstract] [Full Text] [PDF]

				S.-C. Lee, S. Choi, T. Lee, H.-L. Kim, H. Chin, and H.-S. Shin Molecular basis of R-type calcium channels in central amygdala neurons of the mouse PNAS, March 5, 2002; 99(5): 3276 - 3281. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (104) Citing Articles via Google Scholar Google Scholar Articles by Tottene, A. Articles by Pietrobon, D. Search for Related Content PubMed PubMed Citation Articles by Tottene, A. Articles by Pietrobon, D.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help