Gadolinium block of calcium channels: influence of bicarbonate

142

Brain Research, 503 (1991) 142-151~ © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.5t~ A DONIS 0006899391171 l g [!

BRES 17118

Gadolinium block of calcium channels: influence of bicarbonate Linda M. Boland 1'3, Tracy A. Brown 2 and Raymond Dingledine 1'2 1Curriculum in Neurobiology and 2Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (U.S.A.) 3Department of Neurobiology, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 11 June 1991)

Key words: Calcium channel; F-11 cell line; Whole-cell patch clamp; Gadolinium; Dorsal root ganglion; Cardiac muscle; Lanthanide; Bicarbonate

The selectivity of block of voltage-activated barium (Ba2+) currents by lanthanide ions was studied in a rat dorsal root ganglion (DRG) cell line (Fll-B9), rat and frog peripheral neurons, and rat cardiac myocytes using the whole-cell patch clamp technique. Gadolinium (Gd 3+) produced a dose-dependent and complete inhibition of whole-cell Ba2÷ current in all cells studied, including ceils expressing identified dihydropyridine-sensitiveL-type currents and ~o-conotoxin-sensitive N-type currents. Like Gd3+, lutetium (Lu3+) and lanthanum (La3+) blocked all Ba 2÷ current with little selectivity for different components of the whole-cell current. Gd 3÷ block of Ba 2÷ currents was incomplete, however, when sodium bicarbonate (5-22.6 mM) was added to the standard HEPES-buffered external Ba2÷ solution. In rat DRG neurons and F l l - B 9 cells, a fraction of the whole-cell Ba2÷ current recorded in the presence of bicarbonate was resistant to block by saturating concentrations of Gd3+ (50-100 ~M). The resistant current inactivated more rapidly than the original current giving the appearance that, under these conditions, Gd3+ block is more selective for the slowly inactivating component of the whole-cell current. Bicarbonate modification of Gd 3÷ block occurred both before and after w-conotoxin block of N-type currents in rat DRG neurons, suggesting that even in the presence of bicarbonate, Gd 3+ block was not selective for N-type currents.

INTRODUCTION The existence of several types of Ca 2+ channel has been recognized in virtually all excitable cells and has led to the suggestion that Ca 2+ channel subtypes might be specialized for different functions 4'16'18'23'35. Toxins and other drugs that selectively block certain Ca 2+ channel subtypes could help elucidate the role of the different channels. For example, ~o-conotoxin G V I A 1'2°'29, certain spider venoms 24'25, and dihydropyridines 2'17 are useful tools because they block only a fraction of the wholecell current through Ca 2+ channels. Divalent cation blockers, however, have been less useful in differentiating subtypes of Ca 2+ channel because all types of Ca 2+ current 4"11'14'35 can be blocked by Cd 2÷, Ni 2+, Co 2+, and Mn 2+. While different c o m p o n e n t s of the whole-cell Ca 2+ channel current show different potency for block by certain divalents 7'~3'34, none of these blockers is selective enough to be useful in functional studies. The trivalent lanthanide gadolinium ( G d 3+) was rep o r t e d by D o c h e r t y 9 to be a selective blocker of a slowly decaying (r = 800 ms), high-threshold Ca 2÷ current in N G 1 0 8 - 1 5 n e u r o b l a s t o m a x glioma hybrid cells. Do-

cherty r e p o r t e d that a fraction of the whole-cell Ca 2÷ channel current in N G 1 0 8 - 1 5 cells was resistant to block by up to 50/~M G d 3+, and he suggested, based on the kinetic properties of the Gd3+-sensitive c o m p o n e n t , that N-type channels were selectively blocked. In view of the significant implications this finding has for identification and the assignment of functional properties to Ca 2+ channels, we sought to extend this observation. We studied G d 3+ block of Ba 2+ currents in a variety of cell types known to express oJ-conotoxin-sensitive N-type and/or dihydropyridine-sensitive L-type Ca 2÷ currents. Using a H E P E S - b u f f e r e d external solution, we found that G d 3+ blocked all Ca 2+ channel current with little evidence of different potency for different kinetic or pharmacologic components of whole-cell current. In attempting to reconcile our observations with those of D o c h e r t y 9, we found that the presence of sodium bicarbonate dramatically modified the action of G d 3+ by allowing block of only a fraction of the whole-cell Ba 2+ current. E v e n in the presence of bicarbonate, however, G d 3+ block was not selective for N-type current. A preliminary account of this work was presented to the Society for Neuroscience 8.

Correspondence: L.M. Boland, Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, U.S.A. Fax: (1) (617) 734-7557.

143

MATERIALS AND METHODS

Culture of FI I-B9 cells The F l l - B 9 cell line is a clonal strain derived from the F-11 cell line (rat DRG x mouse N18TG-2). F l l - B 9 cells were cultured as previously described7. In brief, F l l - B 9 cells were fed with either a growth medium consisting of Ham's F-12 (Gibco Laboratories, Grand Island, NY) supplemented with 10 mM glucose, 12 mM sodium bicarbonate, 15% fetal calf serum (FCS; Hazelton, Lanexa, KA), and 100/~M hypoxanthine, 0.4 ~M aminopterin, and 16/~M thymidine (HAT; Flow Laboratories, McLean, VA) or a differentiation medium consisting of F-12 with 1% FCS, HAT, 0.5 mM dibutyrl cAMP, 10/~M 3-isobutyl-l-methyl-xanthine, and 50 ng/ml mouse salivary gland NGF (2.5S, Sigma). Experiments were performed on F l l - B 9 cells (passage 2-20) that were plated onto uncoated glass coverslips and fed with differentiation medium for 1-3 days. After this time, many cells grew long, branching processes that prevented acceptable space clamp. Differentiated cells expressed a greater sustained current component than cells grown in growth medium 7.

Preparation of freshly dissociated neurons Patch clamp recordings were made from freshly dissociated DRG or sympathetic ganglion cell bodies to avoid complications associated with inadequate voltage-clamp of neurites. Neurons were dissociated from DRG or sympathetic ganglia from adult bullfrog (Rana catesbiana) or DRG isolated from neonatal rat (P3-P16) according to modifications of the protocol of Huettner and Baughman tg. Ganglia from frog were dissected in an oxygenated Ca z+free Ringer's solution (in mM): NaC1 100, KCI 2.5, MgCI 2 5, glucose 10, HEPES 10, pH 7.4. Ganglia were cut in half, and tissue pieces were incubated at 30-35 °C with 1 mg/ml collagenase (type I, Sigma) plus 5 mg/ml dispase (Boehringer Mannheim Biochemicals, Indianapolis, IN) in the Ca2+-free Ringer's solution. After 1 h, the enzyme solution was replaced with a fresh Ca2+-free Ringer's solution containing 5 mg/ml dispase. The tissue was incubated at room temperature for an additional hour or until trituration yielded single cells. The mixture was then diluted 2-fold with Ringer's solution containing 2 mM CaCI 2 and stored at 4 °C until use. DRG cells from neonatal rats were obtained by a similar protocol except the DRG were dissected in an oxygenated, CaZ+-free Tyrode's solution containing (in mM): NaCI 150, KCI 4, MgC12 2, glucose 10, HEPES 10, pH 7.4. Cells were dissociated by incubation in 20 U/ml papain (Worthington Biochemical, Freehold, NJ) for 1.5 h in Ca2+-free Ringer's solution. The enzyme solution was then replaced by a Tyrode's solution containing 2 mM CaC12, 1 mg/ml trypsin inhibitor (Type II-O, ovomucoid; Sigma), and 1 mg/ml bovine serum albumin (Sigma) at room temperature for at least 15 min until trituration produced single cells. Rat DRG cells were stored in the enzyme inhibitor solution at room temperature until use.

Preparation of freshly dissociated cardiac cells Acutely dissociated cells from adult rat heart were prepared as previously described by Bean and Rios 5. Briefly, after Langendorf perfusion with an oxygenated Ca2+-free saline solution composed of (in raM): NaCI 135, KCI 5.4, MgCI 2 1, NaHPO 4 0.33, HEPES 10, pH 7.4, the heart was peffused for 40 rain with the same solution containing 1.5 mg/ml eollagenase (type I, Wortington) and protease (type XXlII, Sigma) at 37 °C. The heart was then rinsed at room temperature with a solution consisting of (in mM): K +glutamate 140, MgCI 2 5, EGTA 1, glucose 10, HEPES 10, pH 7.4. Atria or ventricles were minced with scissors, triturated, and isolated cells stored in the K+-glutamate solution at 4 °C until use.

Voltage clamp Macroscopic currents through voltage-activated Ca 2+ channels were recorded using the whole-cell configuration of the patch clamp technique ~5. Patch pipettes were made from borosilicate glass tubing (N51A, Drummond Scientific, Broomall, PA or Boralex, Roch-

ester Scientific Co., Rochester, NY), coated with Syigard (Dow Coming Corp.) in some experiments, and fire-polished. Pipettes had resistances of 1-5 Mf~ when filled with internal solution. For experiments with the F l l - B 9 cells, an Axopatch electrometer (Axon Instruments, Burlingame, CA) with a 0.5 Gf~ headstage was used. Currents were filtered at 1 kHz using an 8-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA), digitized at > 2 kHz, and stored on a computer disc. Signals filtered at 20 kHz were stored on a video tape-recorder. For experiments on other cells, a List EPC-7 electrometer (Medical Systems Corp., Greenvale, NY) was used, and currents were filtered with a 10 kHz 4-pole Bessel filter built into the amplifier. As noted in the figure legends, some traces were later filtered at 1 or 2 kHz using an 8-pole Bessel filter (Frequency Devices). Currents were digitized at 5 kHz or 25 kHz (for tail current recordings) and stored on a computer disc. Voltage pulses used to elicit current were 12-200 ms in duration and were applied every 2-20 s. All records were corrected for leak and capacitive currents by substraction of averaged and scaled currents elicited by 4-16 hyperpolarizing pulses (-8 to -20 mV from the holding potential). All experiments were done at room temperature (21-25 °C). Analysis was performed using in-house programs written in C or Basic 23. For F l l - B 9 cells, Ba 2+ currents were recorded with series resistances ranging from 2 to 10 Mf~ (estimated from optimal cancellation of capacity transients), which produced a voltage error of 0.2-1.0 mV for each 100 pA of current. Since Ba 2÷ currents in F l l - B 9 cells were small, recordings were made without series resistance compensation and data points on the current-voltage curves were corrected for the small estimated voltage error induced by the series resistance. In 67 cells fed with differentiation medium, the maximum sustained current was 224 -+ 20 pA and the maximum transient current was 264 +- 24 pA. F l l - B 9 cells selected for electrophysiological study were isolated from other cells, had a roughly spherical cell body about 20-30/~m in diameter, and did not have processes that were longer than the cell diameter. For freshly dissociated neurons, round cell bodies (approx. 10-20 ~m for DRG and 15-30 ~m for sympathetic neurons) were chosen to study and for cardiac muscle, small myocytes (approx. 40/~m in length) were chosen to minimize problems associated with inadequate space clamp. In recordings from freshly dissociated cells, series resistances ranged from 0.5-2.1 Mf~ after compensation (typically 6090%) to produce a voltage error of 0.5-2.1 mV for each 1 nA of current. Only data from freshly dissociated cells with low series resistance and currents small enough to maintain a voltage error of less than 5 mV were analyzed. For all cell types, if there was any sign of inadequate space clamp (regenerative activity, appearance of notches), the experiment was ended. In all recordings, we used holding potentials sufficiently negative to prevent steady-state inactivation of any component of Ca 2+ channel current. F l l - B 9 cells were typically held at -70 or -80 mV, and whole-cell current amplitudes never increased when depolarizations were applied from a holding potential of -110 mV. For all other cells, appropriate holding potentials were determined from the voltage required to prevent Ba 2+ current inactivation in each cell; these were typically -80 to -100 mV for DRG and sympathetic neurons and -90 to -120 mV for cardiac muscle.

Solutions A variety of solutions were used to isolate Ba z+ current through Ca 2÷ channels. For F l l - B 9 cells, the standard internal solution consisted of (in mM): Cs glutamate 100-120, HEPES (Sigma) 40, TEACI 10, BAPTA (Calbiochem) 5, MgCI 2 1, CaC12 0.5, pH 7.37.35, mOsm 305-315. The free Ca z+ concentration was calculated to be less than 15 nM. The standard external solution consisted of (in mM): BaC12 30, TEACI 80 (or TEACI 40 and NaC1 40), glucose 25, CsCI 20, HEPES 5-10, TI'X 0.5-1 ~M, pH 7.3-7.35 with CsOH, osmolality adjusted to 310-320 mOsm with sucrose. Drugs dissolved in the external solution were applied via a perfusion pipette with a large tip opening (20-100/~m) positioned close to the cell.

144 For neurons and cardiac cells, the standard internal solution consisted of (in mM): CsCI 108, HEPES 9, EGTA 9, MgC12 4.5, and an ATP-regenerating solution ~2, pH 7.4 with CsOH. The ATP-regenerating solution consisted of (in mM): creatine phosphate 14 (Tris salt; Sigma), MgATP 4 (Sigma), GTP 0.3 (Tris salt; Bochringer Mannheim or Sigma), and 50 U/ml creatine phosphokinase (Type 1, from rabbit muscle, Sigma), pH 7.4 with Tris base. Thc free Ca 2+ concentration of this internal solution was less than 1 nM and osmolality was 295-305 mOsm. Cells were patched in a modified Tyrode's solution consisting of (in raM): NaCI 150, KCI 4. CaCI 2 2, MgCI 2 2, glucose 10, HEPES 10, and in most cxperiments, BaCI 2 4. Immediately after establishing the whole-cell configuration, cells were lifted off the dish and drugs and external solution were applied by moving the cell in front of a stream of solution applied by a gravity-fed reservoir via one of several microcapillary tubes (1/A 'microcaps'. Drummond Scientific) glued together sideby-side. For freshly dissociated cells, the control external solution consisted of (in mM): BaCI 2 3, 5, or 10, TEACI 160 or 150, HEPES 10, and , in most experiments, TTX 3/*M, pH 7.4 with TEAOH. Other external solutions were used in experiments to characterize the effects of bicarbonate on Gd 3+ block and arc described in the appropriate figure legends. The external solutions contain several common features: TEA and cesium to block K + channels, TI'X to block Na + channels, Ba 2+ as the charge carrier, HEPES to buffer pH, and 5-22.6 mM sodium bicarbonate. All solutions were equilibrated in air to pH 7.3-7.4 with TEAOH or CsOH. Lanthanides, as chloride salts, (99.999% pure, Aldrich Chemical Co., Milwaukee, WI) were diluted into the external solution from concentrated stock solutions. Stock solutions of nimodipine and BayK 8644 (both from Miles Pharmaceuticals, New Haven, CT) were prepared in ethanol or DMSO and were stored in the dark at 4 °C. Working solutions of dihydropyridincs were prepared immediately prior to use by dilution of the stock solution with the external recording solution (final ethanol or DMSO concentrations were ~<0.1%). ~o-Conotoxin fraction GVIA was from Peninsula Peptides (Belmont, CA).

Curve fitting Experimental data were fit to the indicated equation by non-linear least squares regression using the Quasi-Newton minimization method of the commercially available package SYSTAT. Curves were drawn using in-house programs written in Pascal. All data represent means + S.E.M.

c r e a s e t h e local G d 3~ c o n c e n t r a t i o n as well as c o m p e t e w i t h G d 3+ for a c h a n n e l b i n d i n g site. W e w e r e i n t e r e s t e d in d e t e r m i n i n g w h e t h e r s u b m a x i mal concentrations of Gd ~

s h o w e d s e l e c t i v e b l o c k of

kinetically isolated components channel FII-B9

current.

We

o f t h e w h o l e - c e l l C a e,

addressed

this

question

using

cells since t h e w h o l e - c e l l B a 2+ c u r r e n t in t h e s e

cells (see i n s e t Fig. 2 A ) c a n d e s e p a r a t e d i n t o t w o distinct kinetic components,

t r a n s i e n t a n d s u s t a i n e d cur-

r e n t s . In this s t u d y , we h a v e n o t p h a r m a c o l o g i c a l l y sepa r a t e d N - t y p e a n d L - t y p e c u r r e n t s (see b e l o w ) in the F1 l - B 9 cells, a l t h o u g h b o t h c u r r e n t t y p e s a r e e x p r e s s e d 7. A s p r e v i o u s l y d e s c r i b e d 7, t h e t r a n s i e n t a n d s u s t a i n e d c u r r e n t s h a v e d i f f e r e n t i n a c t i v a t i o n r a t e s a n d p e a k activ a t i o n v o l t a g e a n d c a n also b e e x p r e s s e d in r e l a t i v e isolation under certain culture conditions. Transient and s u s t a i n e d c u r r e n t s , h o w e v e r , e x h i b i t e d o n l y m i n o r diff e r e n c e s in s e n s i t i v i t y to b l o c k b y G d ~ .

The dose-de-

p e n d e n c e o f G d 3+ b l o c k o f t h e t w o c u r r e n t s is d e m o n s t r a t e d in Fig. 2. T h e ICso for G d 3~ b l o c k o f t r a n s i e n t c u r r e n t (Fig. 2 A ) was 0 . 9 5 / ~ M a n d for s u s t a i n e d c u r r e n t (Fig. 2 C ) was 0.36 # M . T h u s , G d 3+ b l o c k o f s u s t a i n e d c u r r e n t was o n l y 2 . 6 - f o l d m o r e p o t e n t t h a n b l o c k o f t r a n s i e n t c u r r e n t in F l l - B 9 Current-voltage

cells.

r e l a t i o n s h i p s for t r a n s i e n t (Fig. 2 B )

a n d s u s t a i n e d (Fig. 2D} B a 2+ c u r r e n t s in F l l - B 9

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frog DRG neuron

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RESULTS

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B a z+ c u r r e n t s in all n e u r o n s a n d m u s c l e cells t e s t e d a n d t h e F 1 1 - B 9 cell line. L o w c o n c e n t r a t i o n s o f G d 3+ (300 nM-1/*M)

b l o c k e d w h o l e - c e l l c u r r e n t in frog D R G n e u -

r o n s (Fig. 1 A , n = 3) a n d r a t a t r i a l (Fig. 1C, n = 2) a n d v e n t r i c u l a r (Fig. 1D, n = 5) h e a r t cells. F o r frog symp a t h e t i c n e u r o n s (Fig. 1B, n = 5) a n d r a t D R G

neurons

(n = 10, n o t s h o w n ) , 10 # M G d 3+ b l o c k e d t h e w h o l e cell B a 2+ c u r r e n t . A h i g h e r c o n c e n t r a t i o n o f G d 3÷ (30 ~ M ) w a s r e q u i r e d f o r c o m p l e t e b l o c k o f B a 2÷ c u r r e n t s in t h e F l l - B 9

cells (Fig. 2 A , C ) . T h i s f i n d i n g m i g h t b e

e x p l a i n e d b y t h e h i g h e r c o n c e n t r a t i o n o f t h e c h a r g e carr i e r u s e d for c u r r e n t r e c o r d i n g s in t h e F l l - B 9

cells (30

m M B a 2+) c o m p a r e d to t h e n e u r o n s a n d c a r d i a c m u s c l e cells (3, 5, o r 10 m M B a 2 ÷ ) . H i g h d i v a l e n t c o n c e n t r a tions might screen membrane

s u r f a c e c h a r g e s a n d de-

Fig. 1. Gd 3+ block of Ba 2+ currents in neuronal and cardiac muscle cells. Voltage-activated Ca 2+ channel currents were recorded in external Ba 2+ solutions without (control) or with the indicated concentration of Gd 3+. Currents were recorded from a frog DRG neuron (A), holding potential (Vn) -80 mV, test potential (Vt) -10 mV, a frog sympathetic neuron (B), V n = -100 mV, Vt = 0 mV; a rat atrial cell (C), Vh = -100 mV, Vt = -20 mV; and rat vcntricular cell (D), VI,= -120 mV, Vt = -20 inV. Data in (C) were filtered at 1 kHz, all others were filtered at l0 kHz. The external solution used in recordings from the neurons was (in mM): BaCI z 5, TEAC1 160, HEPES 10, T1FX 0.003, pH 7.4. Myocytes were recorded from in a similar solution containing BaCI 2 10 and TEACI 150.

145 demonstrate that Gd 3÷ did not shift the current-voltage relationships. These data indicate that Gd 3÷ block was complete, and block by submaximal concentrations did not reveal different components of Ca 2+ channel current. Gd 3+ block o f N-type and L-type currents Peripheral neurons have more than one type of Ca 2+ channel 4'16'3s but kinetic, activation, and inactivation properties of Ca 2+ currents are not sufficiently different to justify a kinetic separation of the current components in a whole-cell recording (see control traces Fig. 1A,B). Thus, we directly tested whether pharmacologically defined components of the whole-cell current were selectively blocked or spared by Gd 3+. Since Gd 3+ was reported to be a selective blocker of N-type current 9, we determined whether Gd 3+ block was selective in cells that co-expressed N-type and non-Ntype Ca 2+ channel currents. We compared, in the same cells, Gd 3+ block before and after exposure to a saturating concentration (3/~M) of ~o-conotoxin (CTX) fraction G V I A , a selective blocker of N-type Ca 2+ channels 1'2°'3°. In the present study, 3 MM CTX blocked 51 - 5.3% of whole-cell current in rat D R G neurons (range = 32-78%, n = 9) and 80 --- 6.4% of current in frog sympathetic neurons (range 51-98%, n = 10). Prior to

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Fig. 2. Gd 3+ block of Ba2+ currents in Fll-B9 cells. Fll-B9 cells expressing both transient and sustained Ba2÷ currents were recorded from prior to and after addition of the indicated concentrations of Gd 3+. Currents were separated as described 7 and cumulative dose-response curves for block of (A) transient and (C) sustained currents were constructed. Data points represent means - S.E.M., n = 4-10. Smooth curves in (A) and (C) were fit by non-linear least squares regression to the equation: fractional current = 1/(1 + ([Gd]/ICso)"). For both curves, n = 1.0. Inset in A: Ba2÷ currents recorded with Vh = --70 mV, Vt = +20 mV. Current-voltage curves for (B) transient and (D) sustained currents under control conditions or after block by the indicated, submaxireal concentrations of Gd 3+. Cells were voltage-clamped at -70 mV and all external solutions contained 30 mM BaC12.

or following exposure to CTX, B a 2+ currents in rat D R G and frog sympathetic neurons were blocked completely by 10 MM Gd 3+, indicating that Gd 3+ blocks N-type channels as well as non-N-type channels. It was not possible to completely isolate N-type current by the use of dihydropyridine antagonists to block L-type currents; the remaining current is comprised of both CTXsensitive current as well as a current that is resistant to both CTX and dihydropyridine antagonists 32. All of the cell types tested are known to express L-type dihydropyridine-sensitive Ca 2÷ channels, although the proportion of this current to total current varies considerably in the different cells. Gd 3+ (300 n M - 1 MM) blocked nearly all Ba 2÷ current in rat atrial (Fig. 1C) and ventricular (Fig. 1D) myocytes. Since Ba 2÷ current in cardiac muscle cells is largely L-type current 3, these results suggest that cardiac muscle type L-type channels are blocked by Gd 3+. As a direct test of the possibility that Gd 3+ blocks neuronal L-type Ca 2+ channels, we looked for Gd 3+ block of neuronal dihydropyridine-sensitive current. First, we measured the degree of block of dihydropyridine-enhanced L-type tail current in rat D R G neurons. As described by others 3°'32, dihydropyridine agonists selectively slow the deactivation of L-type Ca 2÷ channels, which is experimentally useful for the separation of L-type and N-type tail current components. In the present study, in the absence of CTX or dihydropyridines, the decay of tail currents in rat D R G neurons was fit well by a single exponential (r = 240-340 Ms). CTX blocked a fraction of the tail current but did not alter the timecourse of decay of the current. After exposure to 1 MM BayK 8644, tail currents decayed more slowly and a second exponential (r = 1.5-3.0 ms, n = 3) was required to fit the timecourse of decay of the tails. Gd 3÷ (10 MM) blocked all Ba 2÷ current during the depolarizing step and also blocked the BayK 8644-slowed tail current during the repolarizing step (n = 3), indicating that Gd 3÷ blocks neuronal L-type current. F l l - B 9 cells also express L-type current 7. To investigate the block by Gd 3+ of isolated L-type current in F l l - B 9 cells, we measured the degree of block by successive applications of 10 MM Gd 3÷ and 300 nM nimodipine to the same cell at a holding potential of -35 mV (n = 8). U n d e r these conditions, all nimodipine-sensitive, L-type current was blocked by Gd 3÷. In summary, G d 3+ completely blocks whole-cell Ba 2+ current through multiple types of Ca 2÷ channel and shows little selectivity for the different pharmacological components of the whole-cell current. Effects of other lanthanides on Ba 2+ currents Two other trivalent cations, lutetium (Lu 3+) and lan-

146

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Fig. 3. Dose-response relationships for lanthanum and lutetium block. Cumulative dose-response curves for (A) lanthanum (La 3+) and (B) lutetium (Lu 3+) block of transient (solid circles) and sustained (open circles) Ba 2+ currents in F l l - B 9 cells. Data points represent means -+ S.E.M. (n = 3-11 cells) and are expressed as a fraction of the maximum transient or sustained component of the Ba 2+ current. Smooth curves were fit as described in the legend to Fig. 2; n = 1.0 for all curves expect for Lu 3+ block of sustained current for which n = 0.9.

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controt

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test potential (mY)

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30

test potential (mV)

Fig. 4. Bicarbonate modifies Gd 3+ block in F l l - B 9 cells. F l l - B 9 cells expressing both transient and sustained Ba 2÷ currents were recorded from prior to and after addition of the indicated concentrations of Gd 3+ in external solution containing sodium bicarbonate 5 raM, HEPES 5 mM and BaC12 20 mM. Cumulative dose-response curves for block of (A) transient and (C) sustained currents. Data points represent means --- S.E.M., n -- 4-10. Smooth curves in (A) and (C) were fit as described in Fig. 2; for the transient current, n - 0.8 and for the sustained current n = 0.9. Inset in A: Ba 2÷ currents recorded with Vh = -70 mV and Vt = +20 mV. Current-voltage curves in the presence of 5 mM sodium bicarbonate for (B) transient and (D) sustained currents under control conditions (HCO 3- alone) or after incomplete block by high concentrations of Gd 3+.

the l a n t h a n i d e s w e r e slightly m o r e p o t e n t b l o c k e r s of sustained c u r r e n t t h a n transient current. L a 3+ d e m o n strated the g r e a t e s t

selectivity, a p p r o x i m a t e l y

12-fold

t h a n u m (La 3+) also c o m p l e t e l y b l o c k e d t r a n s i e n t and

m o r e p o t e n t b l o c k of s u s t a i n e d c u r r e n t t h a n transient

sustained

c u r r e n t in F l l - B 9

B a 2+ c u r r e n t s

in F l l - B 9

b l o c k e d all B a 2÷ c u r r e n t in rat D R G

cells and

La 3+

and frog s y m p a -

cells. A l l fitted curves had slope co-

efficients not significantly d i f f e r e n t f r o m 1.0. This indi-

thetic n e u r o n s . T h e d o s e - d e p e n d e n c e of L a 3+ and L u 3+

cates that, in t h e F l l - B 9

block o f t r a n s i e n t and sustained c u r r e n t s in F l l - B 9

c u r r e n t s are distinguished p o o r l y by selectivity to a par-

cells

is s h o w n in Fig. 3. T h e ICs0s for b l o c k of F l l - B 9 cell B a 2+ c u r r e n t s by G d 3+, La 3+, and L u 3+ are c o m p a r e d in Table I. All of

ticular l a n t h a n i d e .

cells, the kinetically s e p a r a t e d

G e n e r a l l y , block by l a n t h a n i d e s of

B a 2÷ currents was reversible. This was the case for short applications at c o n c e n t r a t i o n s less t h a n 10 # M , h o w e v e r , l a n t h a n i d e applications l o n g e r t h a n 2 min s e v e r e l y ret a r d e d the r e c o v e r y of the B a 2+ c u r r e n t s and the lifetime of the recording. R e v e r s i b i l i t y of b l o c k was i m p r o v e d by

TABLE I

washing the cell with c o n t r o l solution c o n t a i n i n g 0.1 m M

Lanthanide block of Bad+ currents in Fll-B9 cells

EGTA.

ICs0s were determined by best fits to the equation: fractional current = 1/(l+([lanthanide]/ICso) n) with n = 1.0, n = 0.8 (b), or n = 0.9 (c). The maximum block was estimated to be 61% a or 100% in all other cases.

Lanthanide

Gadolinium Gadolinium + 5 mM bicarbonate Lanthanum " Lutetium

lCso (#M) Sustained

Transieht

0.36 0.03" 0.14 0.31c

0.95 67b 1.7 1.3

G ~ + block in the presence o f bicarbonate In an a t t e m p t to r e p l i c a t e the o b s e r v a t i o n s of D o c h e r t y 9, we i n v e s t i g a t e d the characteristics of G d 3+ b l o c k of B a 2+ c u r r e n t s w h e n s o d i u m b i c a r b o n a t e was a d d e d to the s t a n d a r d H E P E S - b u f f e r e d e x t e r n a l solution. T h e addition of s o d i u m b i c a r b o n a t e ( 5 - 2 2 . 6 m M ) r e d u c e d the p r o p o r t i o n of B a 2+ c u r r e n t that could be b l o c k e d by G d 3+. F u r t h e r m o r e , the fraction of B a 2÷ c u r r e n t that was resistant to b l o c k by this m i x t u r e e x h i b i t e d m o r e

147 rapid decay than the original whole-cell current. F11-B9 cells. In the F l l - B 9 cells, where transient and sustained currents can be s e p a r a t e d , the presence of sodium b i c a r b o n a t e (5 m M ) p r o d u c e d an a p p a r e n t decrease in the effectiveness of G d 3+ as a transient current blocker; the IC50 increased from 0.95 to' 67 /~M (Fig. 4A). In contrast, b i c a r b o n a t e p r o d u c e d an a p p a r e n t increase in the potency of block of sustained current by Gd3+; the IC50 decreased from 0.36 to 0.03 # M (Fig. 4C). A s shown in Fig. 4 A , C , bicarbonate did not simply shift the d o s e - r e s p o n s e curve for G d 3+ block. Instead, bicarbonate r e n d e r e d a fraction of the current resistant to block by the highest soluble concentrations of G d 3+ (100/~M). The resistant fraction was comprised of both transient and sustained components. G e n e r a l l y , one-half of the transient current and one-third of the sustained current were not blocked by 100/~M G d 3+ in the presence of b i c a r b o n a t e (Fig. 4 A , C ) . Peripheral neurons. B i c a r b o n a t e caused a similar reduction in the effectiveness of G d 3+ block of Ba 2÷ current in rat D R G neurons. In neurons perfused with a bicarbonate-containing external solution, G d 3+, at concentrations up to 50/~M, only partially b l o c k e d the Ba e÷ current. In the presence of sodium b i c a r b o n a t e (20-22.6 mM), the incomplete block by G d 3+ revealed a substantial transient c o m p o n e n t in all cells and a smaller sustained c o m p o n e n t which was not present in all cells (Fig. 5 A , C ) . F o r e x a m p l e , in the presence of 10 ktM G d 3+ plus b i c a r b o n a t e (n = 9), 39 ± 12% of the p e a k and 11 ± 2.2% of the long-lasting current (measured at 109-119 ms) remained. In contrast, in the absence of bicarbonate only 2.3 _+ 1.0% of the p e a k and 1.5 ± 0.8% of the long-lasting current r e m a i n e d after block by 10 ~ M G d 3÷ (n = 12). The current that was resistant to block by 5 0 100 ~ M G d 3+ in the presence of bicarbonate was nearly eliminated by 300 # M Cd 2÷ (87 _+ 3.6% block, n = 8 F 1 1 - B 9 cells) or 3 m M Cd 2÷ (Fig. 5C; 86 +-- 9.5% block, n = 3 rat D R G neurons). This suggests that a large fraction of these currents were Ca 2+ channel currents. In similar experiments on frog sympathetic neurons, an effect of b i c a r b o n a t e on G d 3÷ block of Ba 2+ currents was less p r o n o u n c e d although block was still incomplete. In the presence of 10 # M G d 3+ plus sodium bicarbonate (20-22.6 mM; n = 7), 13 ± 3.8% of the p e a k and 6.0 --2.0% of the long-lasting current r e m a i n e d in these cells. In contrast, in the absence of b i c a r b o n a t e only 1.3 ± 0.5% of the p e a k and 0.7 ± 0.5% of the long-lasting current r e m a i n e d after block by 10 # M G d 3+ (Fig. 1B, n = 5). The effect of G d 3÷ plus bicarbonate on whole-cell Ba 2+ current in F l l - B 9 cells (Fig. 4) and rat D R G neurons (Fig. 5A) is similar to that previously described for N G 1 0 8 - 1 5 cells 9 where the effect was attributed to se-

lective block of N-type Ca 2+ channels. This explanation seems unlikely since C T X block of whole-cell Ba 2+ current does not alter the relative p r o p o r t i o n s of inactivating and non-inactivating current components. This is seen clearly in Fig. 5B where C T X b l o c k e d equal fractions of the current whether m e a s u r e d at the p e a k or at the end of the 120 ms depolarization. This is a consistent finding in rat and frog p e r i p h e r a l neurons and sug-

A°

10 ~

Cd/HCO31 Gd/HCO3-

l

l nA 40

ms

B.

I 0 #,M OdlHCO3-

m

1hA

C.

3000 uM Cd 50 Gd/HCO3 -

I

1 nA

Fig. 5. Bicarbonate influences Gd 3+ block in rat DRG neurons. A: Ba2+ currents recorded under control conditions (in mM: BaCI 2 3, NaCI 20, TEACI 150, HEPES 10, TI'X 0.003, after exposure to the same solution plus 22 mM sodium bicarbonate (HCO3-), and after partial block by 1 and 10/~M Gd 3+ in the presence of bicarbonate. Ba 2÷ currents in (B) and (C) were recorded in the presence of 22 mM sodium bicarbonate (HCO3-). Currents in (B) are control current (HCO 3- alone), after block by 3/~M to-CTX, and after block of the remaining current by 10/~M Gd 3+. C: control current (HCO 3- alone), after block by 50 /aM Gd 3+, and after block remaining currents by 3 mM Cd 2+. Currents were recorded with Vh = MOO mV, Vt = -10 mV and were filtered at 1 kHz. All data are representative of 4-10 cells.

148 gests that CTX and Gd 3+ plus bicarbonate do not block identical fractions of the whole-cell current. We directly addressed the question of whether bicarbonate confers a selectivity to Gd 3+ block of N-type or non-N-type currents. In the presence of 20-22.6 mM sodium bicarbonate, we compared Gd 3+ block of Ba 2÷ currents in rat D R G neurons both before and after block of N-type current by a saturating concentration of CTX (3 ¢tM). Gd 3+ block in the presence of bicarbonate was incomplete for both the unfractionated whole-cell current (Fig. 5A) as well as the CTX-resistant (non-N-type) currents (Fig. 5B). There is more block by Gd 3÷ plus bicarbonate than can be accounted for by block of only N-type current (Fig. 5A) which represented approximately 50% of the whole-cell current in the rat D R G neurons used in this study. In the presence of bicarbonate, the CTX-insensitive current was unchanged (116 -+ 13% of control) by 10 or 50 #M Gd 3÷ when measured at the peak but was 77 _ 3.6% blocked when measured at the end of the 120 ms pulse (n = 4). Thus, removal of the N-type current also removed the block of peak current but only slightly reduced the block when measured at the end of the pulse. Bicarbonate itself, in the absence of Gd 3+, did not alter the peak Ba 2+ current either before or after CTX exposure but a small fraction of the block measured at the end of the pulse may be explained by the effects of bicarbonate itself (Fig. 5A). Thus, similar to the finding from F l l - B 9 cells (above) and NG108-15 cells9, Gd 3+ in the presence of bicarbonate demonstrated greater block of a long-lasting component of the whole-cell current in rat D R G neurons. The influence of bicarbonate on Gd 3+ block cannot easily be explained by selective block of either N-type or non-Ntype currents, however. Since Gd 3+ in the presence of bicarbonate blocked only a fraction of the whole-cell Ba 2+ current, we considered the possibility that bicarbonate influenced the current-voltage relationship of the whole-cell current. Even in the presence of bicarbonate, however, Gd 3÷ block never induced more than a -10 mV shift in the current-voltage relationship of either the transient (Fig. 4B) or the sustained (Fig. 4D) current in F l l - B 9 cells or the whole-cell current in rat D R G or frog sympathetic neurons (data not shown). Submaximal concentrations of Gd 3+ appeared to block proportionately at all test potentials examined, up to +40 mV. We considered the possibility that modification of Gd 3÷ block of rat D R G Ba 2+ current in the presence of bicarbonate might be due to a change in external or internal pH. External solutions were buffered with 5-10 mM HEPES and the pH of solutions applied to each cell was measured before and after each experiment with a maximum alkalinization of 0.15 units (typically ~< 0.02

for additions of 5 mM bicarbonate and about 0.1 for additions of 20 mM bicarbonate). These relatively small changes in external pH would not be expected to account for the observations reported here. We also investigated the possibility that external bicarbonate might modify Gd 3+ block by altering internal pH. To test this hypothesis, Gd 3+ block was studied in rat DRG neurons using an internal solution with a strong hydrogen ion buffering capacity comprised of (in mM): Cs HEPES 160, CsCI 10, EGTA 10, and the nucleotide regenerating system. In these cells (n = 3), Gd 3+ block in the absence of bicarbonate was complete and block in the presence of 20 mM bicarbonate was incomplete (data not shown). For example, in the presence of 10 pM Gd 3+ plus bicarbonate, 28 + 6.5% of the peak current and 13 -+ 1.4% of the long-lasting current remained. Thus, a change in internal pH cannot be ruled out directly, but it seems unlikely that this would completely explain the modification of Gd 3+ block by bicarbonate. DISCUSSION The most important finding of this study is that bicarbonate modifies the block of Ba 2÷ currents by Gd 3+ in a variety of cell types. Gd 3+ (1-100/~M) dissolved in a HEPES-buffered solution containing bicarbonate produced an incomplete block of Ba 2÷ currents in F l l - B 9 cells and rat D R G neurons. Bicarbonate appeared to enhance the Gd 3+ block of sustained currents and reduce the block of transient currents. In contrast, bicarbonate did not alter the nearly complete block of Ba 2÷ currents by high concentrations of Cd 2÷ and had smaller effects on Gd 3+ block of Ba 2+ currents in frog sympathetic neurons. The Gd3+-resistant current observed in the presence of bicarbonate is reminiscent of that reported by Docherty 9 under similar experimental conditions. This effect of Gd 3+ in the presence of bicarbonate occurred without a consistent change in external pH. Possible effects on internal pH cannot be ruled out but seem unlikely to account for these data since similar observations were made when a strong pH buffer (Cs HEPES 160 mM) was used in the internal recording solution. Furthermore, bicarbonate alone only slightly increased the rate of decay of Ba 2÷ current in peripheral neurons (Fig. 5A) and even occasionally enhanced the current amplitude in F l l - B 9 cells (data not shown). We considered the possibility that the transient current remaining in high concentrations of Gd 3+ in the presence of Na t bicarbonate might be a TTX-insensitive Na + current. Three lines of evidence argue against this possibility. First, the reversal potential of the resistant current is the same as that of the whole-cell Ba 2+ cur-

149 rent, even in the presence of bicarbonate. Second, a TrX-resistant Na + current in rat D R G neurons is induced by external acidification21 and would not be expected to be activated in the bicarbonate experiments in which external pH was 7.4-7.55. Third, the current that remained in F l l - B 9 cells and rat D R G neurons after block by 50-100/~M Gd 3+, in the presence of bicarbonate, was nearly eliminated by 0.3-3 mM Cd 2+ (Fig. 5C) suggesting that a large fraction of these currents were indeed Ca 2+ channel currents. The transient current that remains in rat D R G neurons after block by 10-100 #M Gd 3+ in the presence of sodium bicarbonate is probably not a T-type Ca 2÷ channel current. The whole-cell Ba 2+ current was not enhanced by holding potentials as negative as -100 mV, and only a small fraction of these cells express T-type current 32. Nevertheless, selective pharmacological agents for T-type currents have not been identified so we cannot rule out this possibility. Considering the nearly complete block by Cd 2÷ in the presence of bicarbonate, it seems unlikely that bicarbonate modifies the Ca 2+ channel itself but rather that it modifies the blocking agent. We were unable to locate any direct information concerning Gd3+-bicarbonate complexes although lanthanides are reported to interact strongly with most biological buffers with the exceptions of HEPES and PIPES 1°. It is clear that bicarbonate is not simply reducing the concentration of free Gd 3+, since bicarbonate influenced the block of F l l - B 9 cell sustained and transient currents in opposite directions. A Gda+-bicarbonate complex, therefore, may be a blocking species with different properties from free Gd 3+. It is interesting that bicarbonate has been shown previously to interact with other substances to change their properties. L-fl-Methylaminoalanine, for example, becomes an N M D A receptor agonist in the presence of bicarbonate 33'36. In addition, L-cysteine is rendered excitotoxic and activates larger currents in a bicarbonate-buffered solution than in a HEPES-buffered solution 29. Docherty9 suggested that (in a bicarbonate-containing solution) Gd 3÷ blocks selectively N-type Ca 2÷ channels in NG108-15 ceils. Our data do not support this interpretation since both CTX-sensitive and insensitive Ba 2+ current in rat D R G were fully blocked by Gd 3÷. A hy-

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pothesis that is consistent with our findings is that the decay of the whole-cell Ba 2÷ current in the presence of Gd 3+ plus bicarbonate is due to a time-dependent block of Ca 2+ channels by a Gda+-bicarbonate complex. Block by a Gda+-bicarbonate complex may be promoted by channel opening, as suggested for Gd 3+ block of L-type channels in a rat pituitary cell line 6. In the absence of bicarbonate, all lanthanides tested blocked all Ba 2÷ current components non-selectively, including N-type and L-type currents. The latter finding is consistent with recent reports that lanthanides block (+) 202-791-enhanced single Ca 2÷ channels in a skeletal muscle cell line 22 and L-type channels in endocrine cells 6. In addition to N-type and L-type currents, Gd 3+, in the absence of bicarbonate, must block other types of Ca 2+ channel current such as a novel neuronal high-threshold current that is resistant to block by both CTX and dihydropyridine antagonists 32. Also, based on the complete block of unfractionated Ba 2+ current in cardiac myocytes, it seems likely that Gd 3÷ blocks cardiac muscle T-type currents 3'26'27. Three conclusions can be drawn from our data. First, in the absence of bicarbonate, Gd 3+ and other lanthanides non-selectively block voltage-activated Ca 2÷ channels. Second, sodium bicarbonate alters Gd 3+ block in a complex fashion; the block of Ba 2+ current is incomplete and the remaining current decays more rapidly than the original whole-cell current. Thus, bicarbonate confers an apparently greater selectivity to Gd 3+ block of non-inactivating or slowly inactivating Ba 2+ currents in certain cell types. Finally, even in the presence of sodium bicarbonate, Gd 3+ blocks both N-type and non-Ntype currents.

Acknowledgements. This work was supported by NIH Grant NS23804 (R.D.), a predoctoral fellowship from the National Institute on Drug Abuse (L.M.B.), and by NIH/HL-35034 to Bruce Bean. We thank Miles Pharmaceuticals for the gifts of nimodipine and BayK 8644. We are grateful to Paul Ceelen for help with the preparation of dissociated neurons, Bruce Bean for helpful advice and the preparation of cardiac muscle cells, and Jim Huettner, Isabelle Mintz, Robert Rosenberg, and Kenton Swartz for helpful comments on the manuscript. We also thank R.J. Docherty for descriptions of his unpublished data and comments on an early version of the manuscript.

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