Characterization of Ca2" channel currents in cultured rat cerebellar granule neurones

2929 Journal of Physiology (1995), 482.3, pp. 493-509 493 Characterization of Ca2" channel currents in cultured rat cerebellar granule neurones Hugh A. Pearson *, Kathy G. Sutton *, Roderick H. Scott t and Annette C. Dolphin t Department of Pharmacology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF and tdepartment of Physiology, St George 's Hospital Medical School, Cranmer Terrace, London SE1 7 ORE, UK 1. High-threshold voltage-gated calcium channel currents (IBa) were studied in cultured rat cerebellar granule neurones using the whole-cell patch clamp technique with 10 mm Ba2+ as the charge carrier. The putative P-type component of whole-cell current was characterized by utilizing the toxin w-agatoxin IVA (w-aga IVA) in combination with other blockers. 2. w-aga IVA (100 nm) inhibited the high voltage-activated (HVA) IBa by 40 9 + 3.4% (n = 27), and the dissociation constant Kd was 2-7 nm. Maximal inhibition occurred within a 2-3 min time course, and was irreversible. The isolated o.)-aga IVA-sensitive current was non-inactivating. 3. w-aga IVA exhibited overlapping selectivity with both N- and L-channel blockers; w-conotoxin GVIA (w-ctx GVIA) (1 JM) and the dihydropyridine (-)-202-709 (1 /M), respectively. Together these toxins reduced the w-aga IVA-sensitive component to just 4.5 + 1P4% (n = 3). Thus only a small proportion of the current can be unequivocally attributed to P-type current. Inhibition of the HVA IBa by wo-aga IA also reduced the proportion of w-aga IVA-sensitive current to 28 0 + 3 2 % (n = 3). 4. Application of w-aga IVA and a synthetic form of funnel-web toxin, N-(7-amino-4- azaheptyl)-l-argininamide (sftx-3.3; 10 /#M), produced an additive block of the HVA 'Ba. Consequently these two toxins do not act on the same channel in cerebellar granule neurones. 5. wo-aga IVA inhibition of low voltage-activated (LVA) IBa was studied in the ND7-23 neuronal cell line. w-aga IVA (100 nm) reduced the LVA current by 41 3 + 3 2% (n = 17) in a fully reversible manner with no shift in the steady-state inactivation of the channel. 6. A component of current insensitive to N-, L- and P-channel blockers remained unclassified in all our studies. This component, and also that remaining following block by w-aga IVA and w)-agaia, exhibited relatively rapid, although incomplete, inactivation compared to the other currents isolated in this study. 7. In conclusion, wo-aga IVA inhibits a component of current in cultured cerebellar granule neurones which overlaps almost completely with that inhibited by L- and N-channel blockers. In addition, a large component of whole-cell current in these neurones still remains unclassified. The activation of voltage-sensitive Ca2+ channels and the many other cellular processes, such as channel activation and subsequent entry of Ca2+ into neurones is an important modulation of enzyme activity. Therefore the elucidation mechanism in controlling neurotransmitter release. Ca2+ is of the various Ca2+ channel subtypes present in neurones is also an intracellular messenger responsible for regulating important for our understanding of neuronal function. * Authors' names are in alphabetical order. t To whom correspondence should be addressed.

494 H. A. Pearson and others J. Phy8iol. 482.3 The initial classification of the heterogeneous population of neuronal voltage-activated Ca2+ channels into three main categories, the low voltage-activated (LVA) or T-type, the high voltage-activated (HVA) or L-type, sensitive to 1,4-dihydropyridines (DHP), and the N-type (Nowycky, Fox & Tsien, 1985) subsequently found to be sensitive to w-conotoxin GVIA (w-ctx GVIA) (Kasai, Aosaki & Fukuda, 1987), does not completely describe the currents in all neurones. The identification of different contributions made by these Ca2+ channel types to the whole-cell current has proven difficult because of overlap in their biophysical and pharmacological characteristics (for review see Scott, Pearson & Dolphin, 1991). More recently, interest has centred on the identification of a fourth Ca2+ channel type thought to be responsible for contributing, in part, to the w)-ctx GVIA- and DHP-insensitive high-threshold Ca2+ conductances found in various CNS neurones. This has been named the P-type channel, after a particularly large component of DHP- and w-ctx GVIA-insensitive conductance observed in Purkinje cells (Llinas, Sugimori, Lin & Cherksey, 1989). The pharmacological tools presently available do not appear to be completely selective, having variable actions on different cell types (Scott et at. 1991). This has led to a search for alternative pharmacological agents with different selectivities that could be instrumental in the 'toxityping' of Ca2+ channel subtypes. Spiders evolving as predators have developed potent toxins that are, in many cases, specifically targeted to interact with the constituent proteins of ion channels. Much interest surrounds their possible use as tools to investigate the functional roles played by voltageactivated Ca2+ channels. Recent evidence suggests that a novel forty-eight amino acid peptide toxin, wo-agatoxin IVA (w-aga IVA), isolated from the venom of the American funnel-web spider, Agelenopsis aperta, is a potent inhibitor of the P-type Ca21 channel (Mintz, Venema, Swiderek, Lee, Bean & Adams, 1992b). This toxin has been shown to inhibit selectively a fraction of current resistant to N- and L-type channel blockers in a variety of central and peripheral neurones. A voltage-dependent recovery of the Ca2+ channel block by the toxin can be elicited by a series of depolarizing prepulses to positive potentials (Mintz, Adams & Bean, 1992a). Another fraction of A. aperta venom, believed to contain an arginine polyamine, termed funnel-web toxin (FTX), has also been reported to act as a selective P-type channel blocker (Lin, Rudy & Llinas, 1990; Llina's, Sugimori, Hillman & Cherksey, 1992). A putative structure for this natural toxin has been suggested, and a synthetic form of FTX, (N-(7-amino-4-azaheptyl)-L-argininamide, also known as arginine polyamine, AP, or sftx-3.3) has been produced. Although sftx-3.3 has been found to have activity similar to FTX in some experimental systems (Cherksey, Sugimori & Llinas, 1991) it has also been found to inhibit differentially the LVA Ca2` currents in dorsal root ganglion (DRG) neurones at a concentration of 10 nm (Scott et al. 1992; Sutton, Dolphin & Scott, 1993a), and, at higher concentrations, to inhibit a large proportion of HVA current in these cells. A second polypeptide toxin isolated from A. aperta, w-agatoxin IA (w-aga IA), has also been used to study Ca2+ currents in cultured rat DRG neurones. This toxin has been shown to act as a potent inhibitor of neuronal LVA currents, as well as L- and N-type components of the HVA Ca2+ channel currents in DRGs (Scott, Dolphin, Bindokas & Adams, 1990). Cerebellar granule neurones play an important functional role in the CNS. They form the largest population of cells in the brain, receiving inputs from incoming fast mossy fibres, and forming important excitatory synaptic connections with Purkinje neurones. We and others (Slesinger & Lansman, 1991; De Waard, Feltz & Bossu, 1991; Pearson, Sutton, Scott & Dolphin, 1993; Pearson & Dolphin, 1993) have characterized L- and N-type calcium channel currents in cerebellar granule neurones. Previous studies in our laboratory have also characterized the different highthreshold Ca2+ channels involved in mediating the release of the neurotransmitter glutamate from these cells in response to depolarization (Huston, Scott & Dolphin, 1990; Huston, Cullen, Sweeney, Pearson, Fazeli & Dolphin, 1993). In this study we have further characterized the calcium channel currents in cerebellar granule neurones using the various available toxins known to inhibit calcium channels. However, because cerebellar granule neurones do not exhibit LVA currents, we have also examined the effect of the P-channel blocker )-Aga IVA on LVA currents in the ND7-23 neuronal cell line (Kobrinsky, Pearson & Dolphin, 1994). A preliminary abstract of some of this work has been published (Sutton, Pearson, Scott & Dolphin, 1993 b). METHODS Granule neurones isolated from the cerebella of decapitated 6-day-old rats were grown in culture as previously described by Huston et al. (1990). Non-neuronal cell proliferation was reduced by the addition of 80 /M fluorodeoxyuridine after 48 h. Cells were used between 7 and 14 days in culture. ND7-23 cells (a novel DRG cell line produced by fusion of mouse neuroblastoma N18Tg2 and DRG neurones from neonatal rat, Suburo et al. 1992) were cultur-ed as described by Wood et al. (1990). The growth medium Leibovitz L-15 (Gibco, UK) was employed, supplemented with penicillin (100 units ml-'), NaHCO3 (3 3 g F'), additional glucose (3 3 g F') and 10% heat-inactivated fetal calf serum (Gibco). Differentiation was initiated after 2 days by substituting with supplemented L-15 medium containing 1 mm dibutyryl camp (Sigma,UK), 0 5% fetal calf serum and 2 ng ml-' nerve growth factor. The cells were plated at a density of 5-10 x 103 cells per 22 mm2 of coverslip on polyornithine and laminin-coated

J 4-Agatoxin-sensitive Ca 2+ currents J. Physiol. 482.3 495 coverslips. The differentiating medium was replaced every 3-4 days and cells were used between 4 and 21 days after differentiation. All cells were incubated at 37 C in humidified air containing 5% CO2 Ca2+ channel currents were recorded from cells using the whole-cell patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). To minimize problems of space clamp control in the ND7-23 cells, electrophysiological recordings were obtained from differentiated cells that had been replated 2-3 h previously (Menon-Johansson & Dolphin, 1992). All recordings of Ca2+ channel currents were performed at room temperature using an Axopatch-IB, -1D or 200A patch clamp amplifier and 4-8 MQ micropipettes manufactured from borosilicate glass capillary tubes (Plowden & Thompson, Stourbridge, UK). To avoid problems with spatial control of currents, cells with short processes, usually on the periphery of the dish, were selected. Cells which exhibited 'all-or-none' current-voltage relationships or a stepwise activation of currents were presumed to be inadequately clamped and were discarded, as were cells where the series resistance was greater than 20 MQ. Recordings were only made from cells where the seal resistance was greater than 8-10 G1Q and the holding current at -80 mv was less than 50 pa. Unless stated, cells were clamped at a holding potential (Vh) of -80 mv and capacity transients were electronically compensated. Series resistance was 15-20 MQ2 and 30-70% series resistance compensation was usually used. Currents were evoked by 100 or 150 ms depolarizing voltage steps (0 05 Hz) to command potentials (Va) ranging from -70 to +70 mv. To measure calcium channel tail currents, 3 5-5 MQ pipettes were used. Cells were held at a potential of -90 mv and step-depolarized to a potential of +20 mv for 20 ms before stepping back to a potential of -50 mv for a further 80 ms, after which the voltage was returned to the holding level. Mean series resistance was 12-5 + 0 9 MQ (n = 10) and 50-70% series resistance compensation was used. In the ND7-23 cells the maximum LVA current was measured at a Vc of -30 mv in cells that did not express HVA current. The maximum inward HVA current in the granule cells was activated at a V0 of +10 mv. Linear leak and residual capacity currents were subtracted on-line using a P/10 subtraction protocol (ten steps, one-tenth of the test pulse, averaged and scaled for each test pulse). Current and voltage records were captured on-line at a digitization rate of 5 khz following filtering of the current record (2 khz, 4-pole Bessel filter) using an IBM PS/2 microcomputer attached to a TL-3 interface board (Axon Instruments, USA). In the case of tail current measurement the digitization rate was increased to 25 khz. Pulse protocols, data capture and analysis of recordings were performed using pclamp software (Axon Instruments). To minimize currents flowing through K+, Na+ and Clchannels, cells were bathed in a solution containing (mm): tetraethylammonium acetate (TEA-Ac), 70; N-methyl-Dglucamine (NMDG), 70; KOH, 3; magnesium acetate, 0'6; glucose, 4; Hepes, 10; 'ITX, 0 001. Barium acetate (10 mm) was used as the charge carrier and the solution was adjusted to ph 7-4 with acetic acid and 320 mosmol F' with sucrose. Patch pipettes for use with the granule cells were filled with a Hepes-EGTA solution used by De Waard et al. (1991), which contained (mm): Hepes, 100; EGTA, 30; CaCl2, 3; MgCl2, 1; K2ATP, 1 (ph 7-2 with CsOH, 320 mosmol 1F' with sucrose). The free Ca2+ concentration of this solution is 9 4 nm and the free Mg2+ concentration is 130 #M. The internal solution used for the ND7-23 cells contained (mm): TEA-Ac, 70; NMDG, 70; CaCl2, 1; EGTA, 10; magnesium acetate, 2; K2ATP, 2; Hepes, 10 (ph 7 2 with acetic acid, 320 mosmol 1-l with sucrose). Drugs were applied by pressure ejection from a blunt micropipette (8-12,um tip diameter) placed 30-80,um from the cell, or by perfusion where stated. For perfusion, drugs were supplied via a gravity feed system with a bath volume of 0 4 ml and a flow rate of approximately 1 ml min-'. The polypeptide toxins wo-aga IVA (48 amino acids) and w-aga IA (66 amino acids) were purified as previously described (Adams, Bindokas, Hasegawa & Venema, 1990; Mintz et al. 1992b) and were a generous gift from Dr M. Adams, University of California, Riverside, USA. Samples (2 nmol) were resuspended as stock 20 /zm solutions in distilled water and stored at -20 C between experiments. Stock solution purity of wo-aga IVA was confirmed by electrospray mass spectrometry, which yielded a single peak with a relative molecular mass of 5197 kda. Test solutions were made immediately before experiments by appropriate dilution of the stock solution into recording medium. The synthetic w-aga IVA was supplied by Mr B. Ramesh (Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, London, UK). The synthesis of sftx-3.3 (N-(7-amino-4-azaheptyl)-Largininamide, formerly abbreviated to AP) has previously been described (Scott et al. 1992) and it was kindly supplied by Drs I. Pullar and G. Timms of Lilly Research Centre Ltd, UK. The (-)-enantiomer of 202-791, provided by Dr U. Riiegg (Sandoz, Basel), was dissolved at 10 mm in 70% ethanol before being diluted in bathing solution prior to use. The final ethanol concentration was < 0-01 % and was found to have no effect on Ca2+ channel currents in these cells. The &,-conotoxin GVIA (Peninsula, Merseyside, UK) was dissolved in water. Results are given as means + S.E.M. and statistical significance was determined by Student's t test, paired or unpaired where appropriate. RESULTS Effect of w-aga IVA on Ca2+ channel currents in cerebellar granule neurones The dose-response curve (Fig. 1A) shows that maximal block of the high-threshold Ca2+ channel current (IBa) was achieved in cerebellar granule neurones at 50 nm w-aga IVA (50 + 1P9%, n = 3), with no further significant inhibition by 100 nm toxin. The dissociation constant (Kd) was 2-7 nm. Application of 100 nm synthetic w-aga IVA, for comparison, produced a similar inhibition of 45'0 + 3 9% (n = 9), and 1 JuM of synthetic w-aga IVA produced no additional block. Therefore, saturating concentrations of 100 nm of the native toxin were used throughout our experiments. The kinetics of block seen at this concentration were slow, being similar to those observed in hippocampal neurones and rat DRG neurones (Mintz et al. 1992a). Current inhibition was usually maximal within 2-3 min, and no recovery from toxin block was observed over a maximum time course of 8-10 min.

496 H. A. Pearson and others J. Physiol. 482.3 A 100 90 _ 80 - -- 70 - X 60 50 - s (9) 0 3 c.o 40 - (3) 0 30 20 - (4) (2) (3) 1 0 0 A l l 0 0-001 0-1 1 10 100 1000 w-aga IVA concentration (nm) E 50 C -250.*.5-80 mv 10 mv -50~~~~~ 100~~~~ -50/ -100 CD url.ag BlcIfVAincrbla rauenuoe b l-g V A, cocnrtoceedneo oaaiaboc fhaiai eeelrgauenuontro0m -250 loc -80-60 -40-20 0 20 40 60 80 Test potential (mv) Figure 1. Block of 'Ba in cerebellar granule neurones by w-aga IVA A, concentration dependence of w)-aga IVA block of HVA IBa in cerebellar granule neurones. Block was determined after maximal inhibition had been achieved (3 min application) using 100 ms pulses from a Vh of -80 mv to a V, of +10 mv every 20 s. Application of toxin-free external solution was made to control for run-down. Points are mean values + S.E.M. The variation observed was probably because the dose-response curve was measured using a number of different batches of cells over a long time period. Due to a limited supply of native toxin, synthetic w)-aga IVA was used at a concentration of 100 nm, for comparison, and 1 /LM (0). The continuous curve is drawn according to the equation 7-85 + max/{1 + (Kd/[toxin])8} where max (maximum inhibition) is 36-97%, Kd is 2-78 nm and s (approximates to the Hill coefficient) is 0'56. B, I-V relationship recorded from a granule neurone held at a Vh of -80 mv showing inhibition of control peak IBa (0) by a 3 min bath application of )-Aga IVA (0). The null potential, at which no net inward or outward current is activated, was unchanged at +70 mv. No LVA currents are seen in these cells. C, inhibition of maximum 'Ba activated from a Vh of -80 mv by a 100 ms depolarizing voltage step command to a activation was determined by fitting a V, of +10 mv in the presence of wi-aga IVA for 3 min. The V% single Boltzmann equation of the form: I= g( V - Vrev)/{1 + exp[-( V - V½act)/lk]} where g is the conductance, k is the slope factor, Vrev is the reversal potential, and V½act is the voltage for 50% inactivation. The V17 under control conditions was 2-6 mv, and following w-aga IVA application this decreased slightly to a V,,½ of 1P5 mv. 101)0 pa

w-agatoxin-sensitive Ca 2+ currents J. Physiol. 482.3 497 The inhibitory action of wo-aga IVA was examined over a range of command potentials from a Vc of -70 to +70 mv. Figure 1 B and C shows the I-V relationship and current traces recorded from a cerebellar granule neurone under control conditions and following inhibition by a 3 min application of w-aga IVA from a puffer pipette. The toxin (100 nm) markedly inhibited IBa' reducing the peak current by 40-9 + 34% (n = 27; P < 0001, paired t test) with a small depolarizing shift in the voltage dependence of channel activation (Fig. 1B). The block of IBa by w-aga IVA measured at the end of the 100 ms voltage step was significantly greater than the inhibition measured at the peak, being reduced by 52-0 + 3-3 % (P < 0 001, paired t test). Previous studies by Mintz et al. (1992a) have shown that block by w-aga IVA can be fully reversed in a voltage- dependent manner by employing a depolarizing prepulse protocol 1 s before the test step. We investigated the effect of this prepulse protocol on the peak IBa recorded from cerebellar granule neurones. Figure 2 shows the time course of IBa amplitude in a cell in which the prepulse protocol was employed both under control conditions and with 100 nm w-aga IVA present in the bath medium (Fig. 2A). Although we demonstrated a component of the whole-cell current that is sensitive to w-aga IVA, in this study we were unable to achieve any voltage-dependent reversal of its block. Rather than restoring the inhibited current to control levels, the pulse train reduced both the blocked current and the control current to a similar extent (15-4+8-2%, n=6, Fig. 2C and 23X2+4-2%, n=11, Fig. 2B, respectively; P= 0 356). A B 10 mv -250 r -80 mv -200 - w-aga IVA / Prepulse 6 CL Q a- Cu -150 F -100 F -50 - C 00#00. AM404"It,ti. \ Control "', -,rr-.p-w, Prepulse co-aga IVA 0 80 160 240 Time (s) 320 400 100 pa 50 ms Figure 2. w-aga IVA block of IBa in cerebellar granule neurones is not reversed using a train of depolarizing prepulses A, time course of decline induced by w-aga IVA on peak IBa activated from a Vh of -80 mv by 100 ms depolarizing step commands to a Vc of +10 mv. Current activated following a train of depolarizing prepulses (0) (Vh,-80 mv, series of ten 60 ms voltage steps to Vc, +130 mv, 1 Hz, 1 s prior to activation of peak inward IBa). There is no relief of the block induced by a 4 min application of wo-aga IVA. B and C, depolarizing prepulses inhibit the peak 'Ba (Vh, -80 mv, 100 ms step depolarization to Vc, +10 mv). Current is reduced both under control conditions (B) and in the presence of w-aga IVA (C).

498 H. A. Pearson and others J. Physiol. 482.3 w-aga IVA inhibition of LVA IBa currents in the ND7-23 cell line w-aga IVA has formerly been shown to act selectively on a component of the HVA calcium channel current in a variety of cells, with no apparent effect on the LVA calcium current (Mintz et al. 1992a). Previous work carried out in our laboratory has characterized the various current components exhibited by cultured ND7-23 neurones, a novel DRG cell line (Kobrinsky et al. 1994). The majority of cells used in that study only exhibited LVA calcium A B -80 mvj -30 mv L I C -0 ṁ it, Control Recovery IVA 100 pa 50 ms C 1-2 Test potential (mv) 1.*0 - a 0-8 a) ir Q.N zy 06 0*4 0 0-2 0.0-0-2 1-120 -100-80 -60-40 -20 0 Holding potential (mv) Figure 3. Block of IBa in ND7-23 neurones by w-aga IVA (100 nm) A, I-V relationship recorded from an ND7-23 neurone held at a Vh of -80 mv, showing inhibition of control IBa (0) by a 2 min bath application of wi-aga IVA (0) and its recovery after 5 min (V) following removal of the pressure-ejection pipette from the bath medium. The null potential was unchanged at +70 mv. No HVA component of current was seen in this cell. B, inhibition of maximum LVA IBa elicited by a 150 ms depolarizing voltage step command from a Vh of -80 mv to a Vc of -30 mv under control conditions, in the presence of w-aga IVA (2 min) and following a 5 min recovery. C, inhibition induced by w-aga IVA (100 nm) does not influence the steady-state inactivation of the ND7-23 neuronal Ca2+ channel. Cells were held at potentials (Vh) between -120 and +10 mv for 10 s prior to activation of a LVA IBa current by a voltage step depolarization to a Vc, of -30 mv. Currents were leak subtracted off-line and were recorded under control conditions (0) and following a 4 min application of )-Aga IVA (to establish maximum inhibition) (0). Values are expressed as a fraction of the Imax seen in each cell (n 5, = means + S.E.M.). Curves were fitted with a single Boltzmann equation of the form I/Imax = [1 + exp ( Vh- h)/k]ft where h is the voltage for 50% inactivation and k is the slope factor. For the fitted control curve, h is -68-7 mv and k is 6-3. For the curve in the presence of wo-aga IVA, h is -70 7 mv and k is 7-7.

w-agatoxin-sensitive Ca 2+ currents J. Physiol. 482.3 499 channel currents, as the HVA currents do not appear until the cells have been differentiated in culture for approximately 2 weeks. Since cerebellar granule neurones do not possess a classical LVA current, the effect of w-aga IVA was examined on LVA currents in this cell line. Figure 3A shows the I-V relationship for a typical LVA current observed in differentiated ND7-23 cells. The threshold for activation of LVA current was approximately -60 mv, reaching a maximum at a Vc of -30 mv. In addition to this, these currents can also be distinguished by their rapid inactivation kinetics, typical of LVA calcium channels (Fig. 3B). The I-V relationship shows the reversible inhibition of the LVA current following a 2 min application of w-aga IVA. The maximum LVA IBa (Vc -30 mv) was reduced by 41P3 + 32% (n= 17). Therefore, in ND7-23 cells, w-aga IVA reversibly inhibits the LVA calcium current in a manner different to that previously observed for HVA currents. We also studied the action of w-aga IVA on the steadystate inactivation of LVA current in ND7-23 neurones. The cells were held for 10 s at potentials ranging successively from -120 to +10 mv prior to a 100 ms step depolarization to a Vc of -30 mv. Figure 3C shows that the LVA IBa recorded from the ND7-23 neurones was virtually abolished by a holding potential Vh of -60 mv. This inactivation at relatively hyperpolarized holding potentials is characteristic of the LVA current (Fox, Nowycky & Tsien, 1987), confirming that it is a LVA IBa that constitutes the predominant component of the wholecell current in these neurones. The steady-state inactivation of the LVA IBa was studied both under control conditions and following application of w-aga IVA. Inhibition by w-aga IVA did not significantly affect the inactivation at any of the holding potentials used, although there was a slight but not statistically significant hyperpolarizing shift in the voltage for 50% steady-state inactivation, from -68-3 + 2-2 mv under control conditions to -73-3 + 2-1 mv (n = 5) in the presence of w-aga IVA. The action of w-aga IVA also remained unchanged when the external charge carrier, 10 mm Ba2+, was replaced with 10 mm Ca2+. The peak LVA ICa was reduced by a similar amount to that for 'Ba, decreasing by 40 7 + 13-9% (n = 4) following a 3 min application of w-aga IVA. Thus the currents in these cells are equally sensitive to the toxin in the presence of either charge carrier. Block by w-aga IVA of the LVA 'Ba in ND7-23 neurones was also unaffected by a burst of depolarizing prepulses. As with the HVA IBa recorded from cultured cerebellar granule neurones, both the control and inhibited current were reduced by a similar extent following the prepulse protocol, decreasing by 33-9 (n = 2) and 26-1 + 12-8% (n = 3), respectively. Specificity of w-aga IVA block of IBa in cerebellar granule neurones w-ctx GVIA is thought to be a selective, irreversible blocker of N-type Ca2+ channel currents (Kasai et al. 1987), and the DHP antagonist (-)-202-791, at 1 /1M, is a selective antagonist of the L-type Ca2+ channel (Hof, Riiegg, Hof & Vogel, 1985). In a previous study we have examined the effect of these antagonists on IBa in cerebellar granule neurones (Pearson et al. 1993). In this study we attempted to classify the w)-aga IVA-sensitive component of wholecell current in cerebellar granule neurones by comparing the fraction of current inhibited by this toxin with those components blocked by w)-ctx GVIA and the DHP antagonist (-)-202-791. Selective application of these three different Ca2+ channel antagonists enabled us to dissect out and compare the N- and L-type current components of the whole-cell current, either prior to or following maximal block of IBa by w-aga IVA. In Fig. 4 the current components inhibited by the sequential application of (-)-202-791, w-ctx GVIA and w-aga IVA (Fig. 4A and D; Protocols 1 and 2) are compared to the reductions in peak IB. observed following inhibition by w-aga IVA prior to the selective application of either (-)-202-791 (Fig. 4B and D; Protocol 3) or w-ctx GVIA (Fig. 4C and D; Protocol 4). Sequential application of (-)-202-791 followed by w-ctx GVIA, each at a concentration of 1 /1M, resulted in the progressive reduction of the peak IBa by 29-1 + 1P4 and 29-3 + 4 5% (n = 3) respectively. However, subsequent application of w-aga IVA (100 nm) was not additive and only produced a further inhibition of 4-5 + 1-4% (n = 3) (Fig. 4A and D; Protocol 1). In the reverse order of application, w-ctx GVIA and (-)-202-791 applied together, following 54-0 + 6-1 % inhibition by wo-aga IVA only produced a further inhibition of 6-1 + 4 4% (n = 3) of the control IBa (Fig. 4D; Protocol 2). To identify whether the specificity of w-aga IVA was overlapping with that of (-)-202-791 or w-ctx GVIA we compared the action of the L- and N-type channel antagonists separately, following an application of w-aga IVA (Protocols 3 and 4). In each case, prior addition of w-aga IVA significantly reduced the effective block of the other antagonist (P < 0 005). From the time courses of the whole-cell Ca2+ channel current (Fig. 4B and C) it can be seen that there is no significant difference in the proportion of w-aga IVA-insensitive current inhibited by either of the two channel blockers. The L- or N-type antagonist only produced an additional inhibition of either 5-7 + 2-0% (Protocol 3; n = 3) or 9 7 + 4 0% (Protocol 4; n = 3), respectively. Furthermore, there was also no change in the proportion of w)-aga IVA-insensitive current inhibited by each of these blockers when applied either together as a simultaneous application (Protocol 2), or separately, as in Protocols 3 and 4. To ensure that a change in voltage due to series resistance error was not responsible

500 H. A. Pearson and others J. Physiol. 482.3 for the apparent inhibition of current by w)-aga IVA, w-ctx GVIA or (-)-202-791, we calculated the change in series resistance error over the course of an experiment for the cells shown in Fig. 4A-C. In each case, at the beginning of the experiment the voltage drop across the series resistance was less than 2 mv. At the end of the experiments, the changes in this voltage drop were 1 10, 0-91 and 0-89 mv for the cells in Fig. 4A-C respectively. These changes in series resistance error are, therefore, not large enough to seriously affect our results. These data suggest that w-aga IVA exhibits a considerable degree of overlapping selectivity in cerebellar granule neurones with both the L- and N-type channel blockers, (-)-202-791 and w-ctx GVIA. Interaction between wo-aga IVA and a DHP agonist on IBa tail currents To clarify further the effects of wo-aga IVA on the calcium currents in these cells, we investigated its effects on calcium channel tail currents in cerebellar granule neurones. To measure tail currents, cells were held at a A -350-300 < 0- -250 C CD -200 o -150 l- -100-50 B -150-125 a -100 -: _75 0 a) a) -50 a. 0 C. Time (s) D Time (s) -250r 100 r CL a- :3 C) a1) a. -200-150 -100-50 200 300 400 Time (s) C o< ao 0 m C) (a a 0-4 C 80 ~ 60 40 20 v?qwq9 ṠI 1 m O<DC zs oex?qt 2s 1 2 3 Protocol number L =1 4 Figure 4. w-aga IVA exhibits overlapping selectivity with both (-)-202-791 and w-ctx GVIA All values represent peak IBa HVA currents recorded from cells stimulated with a 100 ms depolarizing voltage step from a Vh of -80 mv to a Vc of +10 mv at a frequency of 0 05 Hz. A, time course of inhibition on peak control IBa induced by the sequential application of (-)-202-791 (1 /tm), w-ctx GVIA (1 /SM) and w-aga IVA (100 nm). Inset, current traces (from the same cell) illustrate the progressive inhibition of IBa and were taken at points a-d from the time course. Band C, prior application of w-aga IVA greatly reduces the inhibitory effect of both (-)-202-791 and w)-ctx GVIA. D, proportion of peak IBa current inhibited by Ca2+ channel antagonists in cerebellar granule neurones. 0, the proportion of current inhibited by application of &-Aga IVA; Q, the current component inhibited by (-)-202-791; 1, current irreversibly inhibited by w-ctx GVIA; E[, current inhibition by (-)-202-791 together with w-ctx GVIA; [1, the residual component of current left unaffected after addition of the blockers. Current inhibition is expressed as a percentage (means + S.E.M.) of the stable initial control IBa. Additional blockers were only added after the current had achieved a steady state of inhibition (usually 2-3 min). Protocols used were: (1) perfusion of (-)-202-791 followed by sequential application of w-ctx GVIA followed by w-aga IVA (n 3); (2) application of w-aga IVA followed by the simultaneous addition of o-ctx GVIA and = (-)-202-791 together (n 3); (3) cumulative application of w-aga IVA followed by (-)-202-791 = (n = 3); and (4) cumulative application of w-aga IVA followed by w-ctx GVIA (n = 3).

J. Physiol. 482.3 w-agatoxin-sensitive Ca2+ currents 501 potential of -90 mv and step depolarized to a potential of A exp (-t/ri) + A exp (-1/72) + C, +20 mv for 20 ms, after which the membrane potential + 2 was stepped back to -50 mv for a further 80 ms before where A and A are the amplitudes of two decay 2 returning to the holding potential. components having time constants for decay of -r and r2 Decay of the tail current at -50 mv could be fitted by an respectively and C is a constant. The time constants for equation of the form: decay had mean values of 1P38 + 0 18 and 10-70 + 1P26 ms A -500 - (+)-202-791 <L -400-0. (D a' (+)-202-791 (b) - -300- CL - e -200 b -100 B Control (a) 0~~~~~~~~~~~~~~~~~~~~~~~~~~20p / 1o0 ms 0 -E-S9S-*9 9* 0 100 200 300 400 500 600 700 Time (s) C 0-250- (-Aga IVA +s-200- (+)-202-791 ia a-aga IVA and Eu -- > 0 1(+)-202-791 (b) 150 a. E Ca D -ioo -% 0~~~~~~~~~~~~~~~~~~~~~~~~~~~10p ~~~~ -50 ~ ~ ~ ~ ~ ~ ~~~~bcontrol (a) 10p 200 pa 0 200 400 600 800 Time (s) Figure 5. Effect of the DHP agonist (+)-202-791 and w-aga IVA on IBa tail currents in cerebellar granule neurones A, effect of (+)-202-791 on the amplitudes of fast (,r, *) and slow (T2, 0) components of calcium channel tail currents calculated from fits to decay of tail currents as in B. 1 M (+)-202-791 was applied at the bar. B, tail currents taken from the same cell as in A. Continuous lines represent double exponential fits to tail currents using Simplex least-squares minimization. The fitting equation had the form A1exp (-t/t1) + A2exp(-t/T2) + C, where Al is the amplitude, 1 ms after the voltage step to -50 mv, of a rapidly decaying component with a time constant Tj; A2 and T2 are the amplitude and time constant of a slowly decaying component of current and C is the offset. For the control current, decay of the tail was fitted with values for A, of -349-1 pa, for r, of 0 97 ms, for A2 of -7-8 pa, for -r2 of 1 2-2 ms and for C of -2-0 pa. In the presence of (+)-202-791, the fit gave values for A, of -213-0 pa, rl of 1 0 ms, A2 of -52-0 pa, r2 of 13X1 ms and C of -1 9 pa. Fitted lines have been extrapolated back to the beginning of the voltage step. C, effects of &-Aga IVA (100 nm) and (+)-202-791 (1 /M) on the amplitudes of fast (r, m) and slow (r2, 0) components of calcium channel tail currents calculated from fits to decay of tail currents as in D. w)-aga IVA and (+)-202-791 were applied as indicated by the bars. D, tail currents taken from the same cell as in C. Continuous lines represent best fits to the tail current data. For control currents, A, was -114-5 pa; rl, 0-98 ms; A2,-5 3 pa; 2, 6-0 ms and C, -0-24 pa. Following application of both (-Aga IVA and (+)-202-791, A, was -45-0pA; Tr, 0 94; A2,-7-8 pa, 7r2, 6-3 ms and C, -0-41 pa. Currents in the presence of (-Aga IVA alone have been omitted for clarity.

502 H. A. Pear6 son t and others J. Physiol. 482.3 (n = 10) with amplitudes 1 ms after the step to -50 mv of -145-6 + 25-0 and -8-2 + 1P3 pa respectively. The mean offset, C, under these control conditions was -0 33 + 0-31 pa. To investigate the nature of the channel subtypes underlying these tail currents, a DHP agonist, (+)-202-791 (1 /1M), was applied to cells (Fig. 5A and B). Following application of (+)-202-791, the amplitude of the slow component of the tail current increased from -5-8 + 1P4 to -33-7 + 11'5 pa (n = 5; P< 0 01) with no change in the time constant. This increase in the amplitude of the slow component was accompanied by an augmentation of the amplitude of the offset at -50 mv from -0 9 + 0 3 to -4-5 + 0-8 pa. In contrast to these effects, (+)-202-791 inhibited the amplitude of the fast component of the tail current, which declined from a mean control level of -143 + 44 to -81 + 29 pa (P < 0 05) following application of the DHP agonist. These data suggest that L-type channels which slowly deactivate following A sftx-3.3 w-aga IVA 0. c 0 L- 3 C. 0- -150-100 -50 200 400 600 800 1000 Time (s) B 10 mv C 10 mv -80 mv -80 mv sftx-3.3 w-aga IVA Control 100 pa 50 pa 50 ms 50 ms Figure 6. sftx-3.3 exhibits a different selectivity from that of w-aga IVA A, time course of inhibition induced by sftx-3.3 (10 /M) with subsequent addition of w-aga IVA (100 nm). The maximum IBa was activated from a Vh of -80 mv to a Vc of +10 mv every 20 s. B, inhibition of peak 'Ba by application of sftx-3.3 followed by the addition of w-aga IVA. Current traces were taken from the same cell shown in A. C, inhibition by sftx-3.3 of peak IBa is unaffected by prior addition of w-aga IVA.

w-agatoxin-sensitive Ca2+ currents J. Physiol. 482.3 503 repolarization underlie the slow tail component in these cells. Inhibition of the fast component of current by (+)-202-791 may be due in part to a non-specific inhibition of another channel type, since we have previously observed that this compound produces an overall inhibition of the peak current measured at a test potential of +10 mv in these cells under these conditions (Pearson et al. 1993). Application of 100 nm w-aga IVA to cells inhibited the fast component of the tail current decay, reducing its amplitude from -148 + 29 to -110 + 35 pa (n= 5, Fig. 5C; P< 0 05) with no significant effect on the time constant. The slow component amplitude was also inhibited (from -10-6 + 1P7 to -2-8 + 3-9 pa; P< 0 05), again with no effect on the time constant for decay, further suggesting a non-specific action of w-aga IVA on the calcium channel currents in these cells. Following block by w-aga IVA, the effect of 1 /SM (+)-202-791 to enhance the slow component of the tail current was greatly attenuated A -250 w-aga IA -200 _- CL 2- a) :3 Q cds -150 _ -100 _ co-aga IVA -50 _- B 0 L 0 100 200 300 400 500 Time (s) 10 mv C w-aga IA w-aga IVA 1 I~~~~~~~~~~~. -80 mv 600 700 800 10 mv -80 mv (-Aga IA Control 100 pa 50 pa 50 ms 50 ms Figure 7. w-aga IA exhibits overlapping selectivity with c-aga IVA A, time course of inhibition induced by o-aga IA (100 nm) followed by w-aga IVA (100 nm). The peak I. was activated from a Vh of -80 mv to a Vc of +10 mv every 20 s. B, inhibition of peak inward IBa by application of w-aga IA with the subsequent addition of w-aga IVA. Current traces were taken from the same cell shown in A. C, current traces from another cell, in which inhibition by to-aga IA of peak IBa is greatly reduced by prior addition of w-aga IVA.

504 H. A. Pearson and others J. Physiol. 482.3 (Fig. 5C and D). The slow component amplitude increased only slightly from -7-6 + 2-9 to -8-2 + 2-5 pa (n = 3) and did not rise above the control amplitude. However, w-aga IVA did not abolish the effects of (+)-202-791 on the fast component of current, which declined from a value of -66 + 25 pa in the presence of w-aga IVA to -30 5 + 9-8 pa following treatment with (+)-202-791. These data further suggest that w-aga IVA is not selective for P-type channels in these cells and indicates that L-type, DHP-sensitive channels are a major component in its blocking action. Inhibition of IBa by sftx-3.3 and w-aga IVA A synthetic analogue of the polyamine funnel-web spider toxin (FTX), termed sftx-3.3, has also been proposed to act as a selective inhibitor of P-type Ca2+ channel currents in cerebellar Purkinje neurones (Llinas et al. 1989, 1992). The action of sftx-3.3 (10 /SM) was compared with that of w-aga IVA (100 nm) by application of the two toxins sequentially to cerebellar granule neurones. To examine the selectivity of these two blockers, the component of current inhibited by sftx-3.3 was determined both prior to and following block by w-aga IVA. A B C From Fig. 6 it can be seen that the action of sftx-3.3 does not overlap with that of w-aga IVA. There was no significant difference between the degree of block achieved by sftx-3.3 in either protocol. When applied before wo-aga IVA, sftx-3.3 reduced the peak IBa by 25-5 + 3-2% (n = 5; P< 0-001, paired t test; Fig. 6A and B). Similarly, when sftx-3.3 was applied after block by w-aga IVA it reduced the current by 29-3 + 3 3% (n = 4; P< 0 05, paired t test; Fig. 6C). From the absence of overlapping selectivity, it appears that these two toxins (at the concentrations used) do not act on the same channel type in cerebellar granule neurones. w-aga IVA also exhibits overlapping selectivity with e-aga IA for IBa in cerebellar granule neurones Another polypeptide toxin isolated from the venom of A. aperta, w-aga IA, acts as a potent inhibitor of neuronal N, L and LVA components of the whole-cell Ca2" channel current recorded from DRG neurones (Scott et al. 1990). We have studied the action of this toxin on IBa in cerebellar granule neurones and compared it to the effect of w-aga IVA. w-aga IA (100 nm) blocked a proportion of the whole-cell IBa when applied either before or following D w-aga IVA-sensitive current (-)-202-791 -sensitive current w-cgtx-sensitive current Residual current after w-cgtx, (-)-202-791 and w-aga IVA E F G H sftx-3.3-sensitive current w-aga IA-sensitive current Residual current after w-aga IA followed by &)-Aga IVA Residual current after w-aga IVA followed by w-aga IA 50 ms 0 4 I/Imax Figure 8. Toxin-sensitive and -insensitive components of the whole-cell Ca2" channel current recorded in cerebellar granule neurones Traces A-C, E and F represent average difference currents obtained by subtracting the current in the presence of blockers from the stable control current, whereas D, G and H are averages of the residual currents insensitive to block. Each current was normalized with respect to Imax, measured as the peak control current in each cell. Vh was -80 mv, V, was +10 mv (n = 27, 10, 13, 3, 11, 3, 3, 3 for A-H respectively). The inactivation phase of residual currents D, G and H were fitted with a single exponential equation plus a constant (C) of the form: Aexp(-t/r) + C, where A is the initial current amplitude (pa), t is time (ms) and T is the time constant for current inactivation. For D, T was 49-8 + 6'4 ms; for C, T was 28-5 + 3 0 ms; and for H, T was 32-0 + 6-3 ms, representing 52-3 + 11-2 (n = 3), 48-1 + 5.9 (n = 3) and 34-9 + 6-2 % (n = 3) of the current, respectively.

w-agatoxin-sensitive Ca 2± currents J. Physiol. 482.3 505 inhibition by )-Aga IVA (100 nm). However, the fraction of w)-aga IA-sensitive current was significantly (P < 0 01, unpaired t test) reduced by a previous application of w-aga IVA. When applied first, w-aga IA inhibited the maximum IBa by 41P7 + 13-2%, with a subsequent block of 280 + 3-2% (n= 3) by w-aga IVA (Fig. 7A and B). Conversely, block by w-aga IA decreased to only 13-4 + 6-8% of the control IBa following an inhibition of 41P4 + 3 5% (n = 3) by w-aga IVA (Fig. 7C). Thus, there appears to be a considerable degree of overlap in the selectivity of these two toxins for IBa in cerebellar granule neurones. The total proportion of current inhibited by w-aga IA followed by w-aga IVA was 69 + 16% (n = 3), and the total proportion of current inhibited when the toxins were applied in the reverse order was not significantly less (54 + 10%; n = 3), indicating that w-aga IA did not occlude block by w-aga IVA. The various components of the whole-cell current identified using the toxins in this study have been summarized in Fig. 8A-H. The w-aga IVA-sensitive current exhibits relatively little inactivation (Fig. 8A), as do the DHPsensitive (Fig. 8B), w-ctx GVIA-sensitive (Fig. 8C) and w-aga IA-sensitive currents (Fig. 8F). For example, the application of w-aga IVA alone (Fig. 8A) resulted in the inhibition of a current component which exhibited inactivation of only 5.4 + 12-8% (n = 27) over the 100 ms voltage step. However, the residual current following application of the three antagonists w-ctx GVIA, (-)-202-791 and w-aga IVA (Fig. 8D) as well as both of the residual currents remaining following application of )-Aga IA followed by w-aga IVA, or vice versa, have faster rates of inactivation (Fig. 8D, G and H). A single exponential fit to the inactivating phase of the current in Fig. 8G and H yielded similar time constants (T), whose average was 30'3 + 3-2 ms, representing 58%5 + 4-8% of the averaged current (n = 6, VI, = +10 mv) (cf. a value for T of approximately 60 ms for the control IBa in these cells reported by Pearson & Dolphin, 1993). A similar fit to the residual current following N-, L- and P-channel blockers (Fig. 8D) yielded an average time constant (T) of 49*8 + 6-4 ms, representing 52-3 + 11-2% of the averaged current (n = 3, VIK = +10 mv). This was significantly slower than the -r obtained for the currents in Fig. 8 and H (P < 0 05, unpaired t test), although a similar percentage of averaged current remained at the end of the step in each case. DISCUSSION To date, a number of different toxins have been employed to classify the various components of the whole-cell calcium current present in both central and peripheral neurones. The main aims of this classification are to enable cloned calcium channels to be identified with current components in neurones and to subsequently assign appropriate functional roles to those channels whose characteristics are successfully defined. Previous studies on cerebellar granule cell calcium channel currents in our laboratory have identified both a DHP-sensitive, L-type channel that is selectively inhibited by the antagonist (-)-202-791, and a w-ctx GVIA-sensitive, N-type, Ca2+ channel current component that is blocked by elevated internal free Mg2+ (Pearson & Dolphin, 1993; Pearson et al. 1993). All experiments performed here utilized a low internal Mg2+ concentration of 130 /1M, at which N-channels would not be blocked (Pearson & Dolphin, 1993). Although effective at inhibiting two well-defined types of Ca2+ channel components, application of these two antagonists does not result in a complete block of the entire whole-cell current. Consequently, a substantial proportion of the resistant Ca2+ channels remained uncharacterized in these neurones. From studies of other cell types, attempts to classify this resistant current have identified an additional highthreshold current that was first characterized in Purkinje neurones and hence called the P-type current. It has been suggested by different groups that this channel is selectively blocked by the polypeptide toxin w,o-aga IVA (Mintz et al. 1992a, b) or the synthetic polyamine sftx-3.3 (Llinas, Sugimori, Lin & Cherksey, 1989). Recent studies of single channel properties of P-type Ca2+ channels in adult cerebellar Purkinje cells gave three separate conductance values of 9, 14 and 19 ps (110 mm Ba2+) (Usowicz, Sugimori, Cherksey & Llina's, 1992). These conductances are within the range of those values reported for N-type channels. Thus, it is essential for future studies to obtain a definitive pharmacological profile of this new current, as the P-type channel cannot be distinguished solely by its single channel properties. We therefore set out to elucidate the pharmacological and biophysical profile of P-type current in cerebellar granule neurones. Our results indicate that although wo-aga IVA inhibits with high affinity a component of the whole-cell Ca2+ channel current in this cell type, prior application of w-ctx GVIA and (-)-202-791, to block the N- and L-type channels present, almost abolished the w-aga IVA-sensitive current. It therefore appears that it is only a very small proportion of the whole-cell current that can be attributed unequivocally to the P-type channel in these cells. This result conflicts with previous reports, where w-aga IVA was not shown to share any selectivity with either N- or L-type antagonists in a variety of peripheral and central neurones, although cerebellar granule neurones were not examined in these studies (Mintz et al. 1992a, b). However, some overlap in the selectivity of w-aga IVA was reported, where it was found to inhibit weakly the N-type current in bullfrog sympathetic ganglion neurones (Mintz et al. 1992a), although in this case the toxin was considered to act via a slower blocking mechanism than that by which it

506 H. A. Pearson and others J. Physiol. 482.3 acted on P-type current. In addition, Brown and colleagues have recently described a component of w-aga IVAsensitive current recorded from acutely isolated rat neocortical pyramidal neurones that shares, in part, a sensitivity to the L-channel antagonist nifedipine but no sensitivity to wo-ctx GVIA (Brown, Sayer, Schwindt & Crill, 1994). We were unable to determine the precise pharmacological profile of the overlapping specificity of w)-aga IVA with N and/or L-type currents. The resolved amplitude of the current inhibited by w-ctx GVIA and/or (-)-202-791 after initial block by w)-aga IVA was only 5-10 pa. However, in this study the overlap of w-aga IVA appeared to be with both N- and L-type currents. It is thus likely that there is a component of current in cerebellar granule neurones which is not P-type as defined by the properties of the cerebellar Purkinje cell current, but is blocked by w-aga IVA, and also by N- and L-channel blockers. The precise nature of this current remains to be determined. However, it is of interest that, like the N-type calcium channel current in these neurones, the w-aga IVAsensitive component of current is also blocked by elevated internal Mg2+ (Pearson et al. 1993). In our previous study (Pearson et al. 1993) we observed the effect of w-ctx GVIA to be partially reversible. In the present study, when w)-ctx GVIA was applied after or together with w-aga IVA or (-)-202-791, no reversible component to its effect was observed, suggesting that w-ctx GVIA reversibly and partially blocks L-channels in these cells, as previously observed (Williams et al. 1992). The considerable overlap observed between the specificity of w-aga IVA and that of w-ctx GVIA together with (-)-202-791 was similar to the overlap between the currents inhibited by w)-aga IVA and the other polypeptide toxin used here, w)-aga IA. Prior application of w-aga IVA markedly reduced the second, w-aga IAsensitive, current component. Previous work on w-aga IA has shown that this toxin inhibits both N- and L-type calcium channel currents in cultured DRG neurones (Scott et al. 1990). It may be possible that the pharmacological profile obtained with wo-aga IVA together with w-aga IA reflects the fact that o-aga IVA also inhibits some N and L components of the calcium channel current in cerebellar granule neurones. The comparison of w)-aga IVA with the other putative P-type channel antagonist, the polyamine sftx-3.3, revealed a complete lack of overlap in the current component targeted by each of these two blockers. The additive nature of the inhibition achieved with these two toxins would appear to indicate that, rather than sharing a specificity for the P-type channel, these two toxins are, in fact, acting on two separate components of the whole-cell Ca2+ channel current in these cultured neurones. These data support previous findings by Scott et al. (1992), who found that sftx-3.3, when applied at low concentrations (10 nm), selectively inhibits the LVA, T-type current, and at higher concentrations inhibits a large proportion of HVA current in rat DRGs and is therefore not a selective antagonist of P-type current in these neurones. In their recent study, Brown et al. (1994) have demonstrated a partial overlap in the selectivity of w-aga IVA and sftx-3.3; however, in this case the reduction of w-aga IVA block by sftx-3.3 may be due, in part, to the higher concentration of the polyamine (1 mm) that was used in their study. The overlap observed between these two blockers may reflect the non-selective nature of sftx-3.3 inhibition observed at these higher concentrations. In addition to blocking the HVA calcium current, wo-aga IVA also inhibited the current recorded from the ND7-23 neurones. This was the only example of reversible peptide toxin inhibition we encountered in our experiments. Full reversal of toxin block was achieved within 5 min of removing the drug pipette from the bath. These data contrast with the findings of Mintz et al. (1992a) who did not observe any inhibition of the low-threshold current of DRGs and failed to show any significant recovery from toxin block without the aid of a depolarizing prepulse protocol. In an attempt to reverse the w-aga IVA inhibition of the HVA current in the cerebellar granule neurones we applied this same prepulse protocol to currents blocked by w-aga IVA. The return of the current to control levels is thought to represent the actual unbinding of the toxin from the channel, although the mechanism by which depolarization relieves the block still remains unclear. However, in neither cell type used were we able to obtain any recovery of the HVA current that would signify a voltage-dependent reversal of the block by w-aga IVA. The slight decrease in current amplitude recorded both under control conditions and following toxin inhibition was presumed to be due to current inactivation, enhanced by the large positive command potentials used for the prepulses. The mechanism of block observed in these cell types is therefore atypical compared to previous studies, with two exceptions. The previously described slow partial inhibition of w-ctx GVIA-sensitive, N-type channels in bullfrog sympathetic ganglion neurones (Mintz et at. 1992a) was also not relieved by trains of depolarizing prepulses. In addition, Soong, Stea, Hodson, Dubel, Vincent & Snutch (1993), in a study on a cloned Ca2+ channel al-subunit, rbeii, expressed in oocytes, have recently described a partial block of the rbe II current by o-aga IVA (200 nm). A depolarizing prepulse protocol also proved ineffective at removing this inhibition. This reinforces the conclusion that the component of current inhibited by w-aga IVA in cerebellar granule neurones is not the classical P-type, although the block is of high affinity. Another recently described peptide calcium channel antagonist is w-conotoxin MVIIC (w-ctx MVIIC), isolated from the venom of Conus magus. This toxin acts as a highaffinity inhibitor of mammalian presynaptic Ca2+ channels,

J. Physiol. 482.3 wo-agatoxin-sensitive Ca 2+ currents 507 exhibiting overlapping selectivity with wo-ctx GVIA, in addition to blocking P-like current with an IC50 of between 1-10 /M (Hillyard et al. 1992). However, displacement binding assays on rat brain membrane, comparing w-ctx MVIIC with e-aga IVA, revealed that the binding of these toxins is not competitive. It is therefore assumed that these two antagonists either (a) act on different channel types, or (b) bind to the same channel complex at different sites. Ellinor et al. (1993) indicated that w)-ctx MVIIC inhibits an additional component of current in cerebellar granule neurones, which has been termed Q-current (Zhang et at. 1993). The reconciliation of the existing pharmacological classification of Ca2+ channels with the prolific information on their structure emerging from molecular cloning experiments remains a complex issue. We have attempted to place our own pharmacological and biophysical findings within the context of the current classification of cloned Ca2+ channels. In the search to assign the various branches of the Ca2+ channel gene family to the different components of the whole-cell Ca2+ channel current, no clone has yet been found that can be unequivocally associated with the characteristics of the P-type component. The class A axl-subunit (ala) is often compared to the P-type Ca2+ channel. Antibodies specific to portions of the ala sequence interact strongly with cerebellar Purkinje cells but also with terminals synapsing onto their dendrites, suggesting that this calcium channel type is also present in granule neurones (Westenbroek, Hell, Sakurai, Snutch & Catterall, 1993). However, when the ala subunit is expressed in oocytes it generates a Ca2+ channel current with 100-fold less affinity for w-aga IVA (Sather, Tanabe, Zhang, Mori, Adams & Tsien, 1993) than the native P-type channel found in Purkinje neurones (Mintz et al. 1992b). The ala-subunit also exhibits a prominent decay during a 300 ms test pulse, whilst P-type currents typically display a non-decaying waveform. In our study, w-aga IVA also inhibited a relatively non-inactivating component of current, although this is unli4ely to be P-current as originally described. Of the remaining family of Ca2+ channel clones, only one other, the ale, class E clone has been shown to exhibit any sensitivity to w-aga IVA. Experiments on two related clones with similar sequences, from the rat and electric eel, expressed in vitro, have produced differing results. The rbe II clone, isolated from rat brain, was shown to be partially blocked by w-aga IVA (200 nm) in an irreversible manner (Soong et al. 1993); however, the doe-i clone isolated from the forebrain of Discopyge ommata was found to be quite insensitive to w-aga IVA (Ellinor et al. 1993). Our observations in cerebellar granule neurones support the theory that the Ca2+ current resistant to N- and L-type channel blockers in these cells cannot be attributed to P-type channels. Prior studies (Pearson et at. 1993; H. A. Pearson, unpublished observations) have shown that complete block of 'Ba in cerebellar granule cells can be achieved by application of 10-100 /SM Cd2. In our study a Cd2+-sensitive fourth component of current remained resistant even in the combined presence of all types of blockers used (Fig. 8). However, whilst all three of the remaining residual currents isolated in our study appeared to account for approximately 40% of the total IB., the components insensitive to w-aga IVA and w-aga IA exhibited more rapid inactivation kinetics than the residual current remaining following block by w-aga IVA, w-ctx GVIA and (-)-202-791. In a similar study on cerebellar granule neurones cultured in low K+ conditions, Ellinor et al. (1993) have identified a rapidly decaying wi-ctx GVIA-, w-ctx MVIIC-, w-aga IVA- and DHPinsensitive current which they believe may be a mammalian counterpart to the expressed doe-1 channel and have named it R (Zhang et al. 1993). In our study the currents insensitive to the combinations of blockers used also appeared to have a greater rate of inactivation than the other components of current, although this may result from an alteration of channel kinetics by w-aga IA (Scott et al. 1990). In addition, it cannot be ruled out that some of the antagonists used here as selective blockers may bind to, but not inhibit, other Ca2+ channels, while sterically hindering the action of other antagonists, causing incomplete blockade. It is also possible that w-aga IVA may produce partial block of currents corresponding to the A- and E-type clones as has been shown in oocyte expression studies (Sather et al. 1993; Soong et al. 1993). We therefore conclude that although a component of current in cultured cerebellar granule neurones is inhibited by w-aga IVA, this is not P-type as it shows overlapping sensitivity with L- and N-channel blockers. In addition, despite the combined use of antagonists to block all three of the HVA calcium channel-types identified so far, a large component of rapidly inactivating whole-cell current in these cells still remains unclassified. 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