©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Determinants of High Affinity Phenylalkylamine Block of L-type Calcium Channels (*)

(Received for publication, June 12, 1995; and in revised form, July 28, 1995)

Gregory H. Hockerman Barry D. Johnson Todd Scheuer William A. Catterall

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The high affinity phenylalkylamine(-)D888 blocks ion currents through L-type Ca channels containing the alpha subunit with an apparent Kof 50 nM, but N-type Ca channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher K value of 5 µM. L-type Ca channels containing alpha subunits with the site-directed mutations Y1463A, A1467S, or I1470A in the putative transmembrane segment S6 in domain IV (IVS6) were 6-12 times less sensitive to block by(-)D888 than control alpha. Ca channels containing paired combinations of these mutations were even less sensitive to block by(-)D888 than the single mutants, and channels containing all three mutations were >100 times less sensitive to(-)D888 block, similar to N-type Ca channels. In addition, the Y1463A mutant and all combination mutants including the Y1463A mutation had altered ion selectivity, suggesting that Tyr-1463 faces the pore and is involved in ion permeation. Since these three critical amino acid residues are aligned on the same face of the putative IVS6 alpha-helix, we propose that they contribute to a receptor site in the pore that confers a high affinity block of L-type channels by(-)D888.


INTRODUCTION

Voltage-gated Ca channels constitute a family of integral plasma membrane proteins that form highly selective, calcium-conducting pores upon membrane depolarization and thereby couple cell surface electrical signals to intracellular events such as contraction, secretion, and protein phosphorylation (reviewed in Refs. 1 and 2). The pore-forming alpha(1) subunits of voltage-gated Ca channels consist of four homologous domains (I-IV), each containing six putative transmembrane segments (S1-S6)(3) . Voltage-gated Ca channels are blocked by phenylalkylamines, which are thought to bind within the intracellular mouth of the ion-conducting pore(4) . Block of L-type Ca channels in cardiac and smooth muscle by verapamil and related phenylalkylamines is an important therapy for hypertension, cardiac arrhythmias, and angina pectoris(5) . In contrast, N-type Ca channels in neurons are relatively insensitive to block by these drugs.

The phenylalkylamine(-)D888 (desmethoxyverapamil) binds to L-type Ca channels with high affinity (6) and potently blocks L-type currents(7) . Photoaffinity labeling of purified skeletal muscle L-type Ca channels with the high affinity phenylalkylamine LU49888 (8) resulted in highly selective derivatization of a peptide containing transmembrane segment S6 in domain IV (IVS6)(9) . Several lines of evidence have also implicated the S6 segment of K channels (10, 11, 12, 13) and the IVS6 segment of Na channels (14) as components of the binding sites for intracellular pore-blocking drugs. These findings led us to investigate the role of segment IVS6 in high affinity phenylalkylamine block of L-type Ca channels. We report here that three amino acid residues in segment IVS6, Tyr-1463, Ala-1467, and Ile-1470, are required for high affinity block of L-type Ca channels by the phenylalkylamine(-)D888 and are also implicated as pore-lining residues in the intracellular mouth of the pore.


EXPERIMENTAL PROCEDURES

Construction of Mutants

All mutations were constructed using oligonucleotide-directed mutagenesis as described previously (15) . The 1.5-kilobase EspI fragment of the alpha subunit of rat brain Ca channels (16) was subcloned into the bacteriophage M13 mp19 for recovery of single-stranded DNA template. Mutations were inserted into full-length channel constructs in the expression vector Zem 229 (Dr. Eileen Mulvihill, Zymogenetics Corp., Seattle, WA) using the 272-base pair DraIII fragment (nucleotides 4349-4620). All mutations were confirmed by DNA sequencing.

Cell Culture

tsA201 cells, a subclone of the human embryonic kidney cell line HEK293 that expresses SV40 T antigen (a gift of Dr. Robert Dubridge, Cell Genesis, Foster City, CA), were maintained in monolayer culture in Dulbecco's modified Eagle's medium/F-12 medium (Life Technologies, Inc.), supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), and incubated at 37 °C in 10% CO(2). PC12 cells, a rat pheochromocytoma cell line (17) that differentiates into sympathetic neurons in nerve growth factor (NGF), (^1)were grown in the same medium and were exposed to NGF (50 ng/ml) for 7 days prior to recording. Only cells showing a rounded morphology and neurites at this time were used to record N-type currents. All data in PC12 cells were obtained in the presence of 1 µM isradipine (±PN200-110) to block L-type Ca current in PC12 cells. The largest block of the total current measured upon addition of isradipine was 5%, and in most cases no block was detected (n = 5).

Expression

Wild type and mutant rat brain alpha channel subunits (16) were expressed with beta subunits (18) in the pMT-2 vector (Genetics Institute, Cambridge, MA) and alpha(2)(1) subunits (19) in the Zem 228 vector (Dr. Eileen Mulvihill, Zymogenetics Corp.). cDNAs encoding these channel subunits and the CD8 antigen (EBO-pCD-Leu2, American Type Culture Collection) were transfected into tsA-201 cells by CaPO(4) precipitation as described(20) . tsA201 cells, 75% confluent in 35-mm dishes, were transfected with a total of 4 µg of DNA containing an equimolar ratio of the three channel subunit cDNAs and 0.8 µg of CD8 cDNA. After addition of DNA, cells were incubated overnight at 37 °C in 5% CO(2). Twenty hours after transfection, the cells were removed from the culture dishes using 2 mM EDTA in phosphate-buffered saline and replated at low density for electrophysiological analysis. Transfectants were selected by fluorescent antibody labeling (phycoerythrin-labeled anti-CD8, Sigma) using an epifluorescent microscope (Nikon Diaphot, rhodamine filters).

Electrophysiology

Barium currents through L-type Ca channels were recorded using the whole-cell configuration of the patch clamp technique. Patch electrodes were pulled from micropipettes (Van Waters & Rogers) and fire-polished to produce an inner tip diameter of 4-6 µm. Currents were recorded using a List EPC-7 patch clamp amplifier and filtered at 2 kHz (8-pole Bessel filter, -3 db). Data were acquired using Basic-Fastlab software (Indec Systems). Voltagedependent currents have been corrected for leak using an on-line P/4 subtraction paradigm.(-)D888 was applied to cells by addition of 0.5 ml of a 3times stock to a 1-ml bath. The extracellular (bath) saline contained 150 mM Tris, 4 mM MgCl(2), 10 mM BaCl(2), and pH adjusted to 7.3 with methanesulfonic acid. In one set of experiments, N-methyl-D-glucamine (150 mM) was substituted for Tris in this extracellular saline. Patch electrode saline (intracellular) contained 130 mMN-methyl-D-glucamine, 10 mM EGTA, 60 mM HEPES, 2 mM MgATP, 1 mM MgCl(2), and pH adjusted to 7.3 with methanesulfonic acid. All experiments were performed at room temperature (20-23 °C). No nonlinear outward currents were detected under these conditions.


RESULTS AND DISCUSSION

Block of Wild-type L-type and N-type Calcium Channels by (-)D888

The L-type Ca channel alpha subunit (16) was expressed in tsA201 cells (20) together with the beta(18) and alpha(2)(1)(19) subunits. Barium currents through the resulting L-type Ca channels were blocked by(-)D888; a concentration of 50 nM(-)D888 reduced the barium current by approximately 50% (Fig. 1A). The block by (-)D888 was rapid and reached equilibrium within 200 s (Fig. 2A). Analysis of equilibrium block of barium currents by a range of concentrations of(-)D888 yielded an IC of 48 ± 5 nM (Fig. 2B).


Figure 1: Block of wild-type and mutant Ca channels by(-)D888. L-type barium currents were recorded from wild-type alpha (A), A1467S,I70A (B), and Y1463A,A67S,I70A (C), and N-type barium currents were recorded from alpha in a PC12 cell in the presence of 1 µM isradipine as described under ``Experimental Procedures'' (D). Examples of barium current records from individual cells are presented in which an ascending series of doses of(-)D888 was applied. Currents were recorded during 100-ms depolarizations to +10 mV from a holding potential of -60 mV.




Figure 2: Reduced affinity for block of mutant Ca channels by(-)D888. A, time course of(-)D888 block of Ca channel current in wild-type alpha and Y1463A,A67S,I70A triple mutant. (-)D888 was applied at the dose and time indicated while monitoring the barium current at 10-s intervals. Currents were recorded as shown in Fig. 1during 100-ms depolarizations to +10 mV from a holding potential of -60 mV. Examples are from individual cells representative of eight experiments for control (squares) and six for YAI (circles). B, dose-response curves for (-)D888 block of wild-type alpha (circles), I1470A (triangles), Y1463A,A67S,I70A (squares), and N-type current in PC12 cells (diamonds). Errorbars represent standard error (wild-type, n = 5-18; I1470A, n = 1-5; YAI, n = 3-6; N-type, n = 2-4). Smoothlines represent fits to the mean data for the equation, block = 100/(1 + (IC/[D888])) and had the following values: wild-type, IC = 47 nM, h = 0.96; I1470A, IC = 292 nM, h = 1.02; YAI, IC = 5.1 µM, h = 1.57; N-type, IC = 5.4 µM, h = 1.32.



In contrast to the results with L-type Ca channels containing alpha, the N-type Ca channels in the PC12 pheochromocytoma cell line were unaffected by 500 nM(-)D888 (Fig. 1D). A concentration of 5 µM(-)D888 was required to reduce the peak barium current by approximately 50%. Analysis of block by a range of concentrations of(-)D888 indicates that N-type Ca channels have an IC of 5.4 ± 0.8 µM for(-)D888 (Fig. 2B).

Effects of Mutations in Transmembrane Segment IVS6 of the alpha(1)Subunit

The putative transmembrane segment IVS6 of the alpha subunit of L-type Ca channels contains primarily hydrophobic amino acid residues (Fig. 3A). Of the 21 amino acid residues predicted to comprise this transmembrane segment, only 6 are different in the phenylalkylamine-insensitive N-type Ca channels (Fig. 3A). In order to assess the role of the individual amino acid residues in the IVS6 segment of alpha in high affinity block by(-)D888, we mutated alanine 1467 to serine as in N-type Ca channels (Fig. 3A) and the other amino acids in IVS6 to alanine and screened the mutant channels for sensitivity to(-)D888. Alanine was chosen for substitution because it has minimal effects on protein secondary structure (21) and therefore is expected to reduce the hydrophobicity and size of the amino acid residue in each position in this putative alpha helix without causing global conformational change. Some of these single amino acid mutations caused marked increases in the IC values for block of barium currents by(-)D888. For example, the mutation I1470A caused approximately a 6-fold rightward shift in the concentration dependence of block by(-)D888 (Fig. 2B). The IC values for block of barium currents through the wild type and all of the mutant alpha subunits are illustrated in Fig. 3B. Of the 17-amino acid substitutions studied, only Y1463A (IC = 593 ± 220 nM), A1467S (IC = 556 ± 60 nM), and I1470A (IC = 292 ± 1 nM) caused significant increases in the IC for(-)D888 block (Fig. 3B). These results suggest that these three amino acid residues may contribute to formation of the high affinity receptor site for(-)D888 and other phenylalkylamines.


Figure 3: Non-conserved amino acids in segment IVS6 contribute to the high affinity(-)D888 binding site in alpha. A, sequence alignment of channel IVS6 segments. The amino acid sequences of IVS6 transmembrane segments from three ion channels are compared. Residues in alpha (rat brain N-type Ca channel) and alpha (rat brain type IIA Na channel) that differ from those in alpha (rat brain L-type Ca channel) are indicated. Blanks indicate identical residues. Asterisks indicate positions in IVS6 that are not conserved between alpha and alpha calcium channels and are critical for binding of local anesthetics in alpha. B, effect of IVS6 mutations in alpha on block by(-)D888.(-)D888 concentrations ranging from 5 nM to 50 µM were applied to tsA-201 cells expressing alpha channels with mutations in IVS6(16) . The resulting channel block data were fitted with the equation block = 1/(1 + (IC/[D888])) to give the IC values shown (±S.E.; n = 38 for control, n = 1-17 for mutants). Ca channel current (carried by 10 mM Ba) was monitored once every 10 s by a 100-ms depolarization to +10 mV from a holding potential of -60 mV. Bar labeled N-type indicates IC for Ca channel current recorded from NGF-differentiated PC12 cells that express N-type Ca channels containing alpha.



Additive Effects of Multiple Mutations in Transmembrane Segment IVS6

If these three amino acid residues are the primary determinants of high affinity binding of(-)D888, mutation of combinations of them should increase the apparent K(d) for block of the L-type Ca channels to a value near 5 µM like the N-type Ca channels. The double mutation A1467S,I70A (AI) required nearly 500 nM(-)D888 for half-maximal block (Fig. 1B). The IC for(-)D888 block was increased to a mean value of 1.1 ± 0.4 µM (Fig. 3B). The double mutants Y1463A,A67S (YA) and Y1463A,I70A (YI) were even less sensitive to(-)D888 with IC values of 4.0 ± 0.7 and 3.6 ± 1.1 µM, respectively (Fig. 3B). The triple mutation Y1463A,A67S,I70A (YAI) caused an even further increase in the IC for block by(-)D888. Approximately 5 µM(-)D888 was required to give half-maximal block of this mutant L-type Ca channel (Fig. 1C). Even though the affinity for(-)D888 was markedly reduced, block of the mutant YAI reached equilibrium within 200 s at each of the concentrations tested (Fig. 2A). Analysis of the block of barium currents by a range of concentrations of(-)D888 at equilibrium yielded an IC of 5.0 ± 0.8 µM (Fig. 2B). This triple mutation caused an increase in IC for block by(-)D888 that was comparable with those for the double mutants YA and YI (Fig. 3B).

Each concentration of drug tested in these experiments reached an equilibrium level of block with mutant YAI (Fig. 2A) and with the other mutants studied, indicating that the changes in IC reflect changes in the equilibrium K(d) for drug binding. The changes in free energy of binding of(-)D888 caused by each mutation (Delta(DeltaG)) can be estimated from the measured K(d) values according to the equation, Delta(DeltaG) = -RTln(K(d)/K(d)). For the single mutants with significant effects on(-)D888 binding, the Delta(DeltaG) values were: Y1463A, 1.5 kcal/mol; A1467S, 1.4 kcal/mol; and I1470A, 1.1 kcal/mol. For double mutants, the Delta(DeltaG) values were: YA, 2.6 kcal/mol; YI, 2.5 kcal/mol; and AI, 1.8 kcal/mol. For the triple mutant YAI, the Delta(DeltaG) value was 2.7 kcal/mol. The Delta(DeltaG) value for the mutation Y1463A was approximately additive with those of the mutations A1467S or I1470A in double mutants YA and YI, but Delta(DeltaG) values for mutations A1467S and I1470A were less than additive in double mutant AI or in the triple mutant. These changes in K(d) values imply that the reductions in binding free energy (Delta(DeltaG)) for(-)D888 are approximately additive for the combined mutation of Tyr-1463 and either Ala-1467 or Ile-1470 but are less than additive for the combined mutation of Ala-1467 and Ile-1470.

Comparison with N-type CaChannels

N-type Ca channels containing the alpha Ca channel subunit in PC12 cells (22, 23) are also >100 times less sensitive to block by(-)D888 (Fig. 1D, Fig. 2B, and Fig. 3B). An alignment of the IVS6 segments of alpha and alpha shows that Tyr-1463, Ala-1467, and Ile-1470 are substituted with Ile, Ser, and Met, respectively, in the alpha subunit (Fig. 3A). To determine whether these amino acid changes could contribute to the difference in sensitivity of L-type and N-type channels to(-)D888 block, the alpha mutant Y1463I,A67S,I70M was constructed containing these three amino acid changes (YAI(N)).(-)D888 blocked this mutant with an IC of 5.4 ± 0.8 µM, not significantly different from the native N-type channels or mutant YAI (Fig. 3B), suggesting that these molecular differences may contribute to the low affinity of N-type channels for(-)D888. Because the amino acid sequences of the alpha(1) subunits of N-type and L-type calcium channels are only about 40% identical(22) , other structural differences between the two channels may also prevent high affinity binding of phenylalkylamines. Therefore, substitution of Tyr-1463, Ala-1467, and Ile-1470 for the corresponding amino acids in IVS6 of the N-type channel may not be sufficient to confer high affinity(-)D888 block.

Functional Properties of Mutant YAI

To examine the specificity of the effects of the triple mutation YAI on Ca channel function, we compared its kinetic and voltage-dependent properties to those of Ca channels containing wild-type alpha The voltage dependence of channel activation was shifted approximately 8 mV more positive in the mutant YAI compared with wt alpha (wt, V = -1.6 ± 2.0 mV; YAI, V = 6.4 ± 2.5 mV) (Fig. 4, A and B). Block by(-)D888 did not alter this relationship between mutant and wt for the voltage dependence of activation (Fig. 4, A and B). Voltage shifts were not due to differences in the time from forming the whole-cell patch clamp configuration since current-voltage relations were first measured at 5 min after break-in, and no additional shifts were observed after that time.


Figure 4: Functional properties and location of critical residues. Current-voltage relations of peak Ca channel current in the absence (circles) and presence (squares) of(-)D888 for control (A) and Y1463A,A67S,I70A triple mutant (B). Mean and standard error are shown (control alpha and 50 nM(-)D888, n = 10; YAI control and 5 µM (-)D888, n = 5). Data for each cell were normalized by dividing the measured current at each potential by the peak current in the absence of drug before averaging. Apparent reversal potentials were estimated by linear extrapolation of the data between +20 and +40 mV to the abscissa. C, dependence of block on holding potential. Test depolarizations to +10 mV (100-ms duration) were preceded by a 5-s conditioning pulse to the indicated holding potentials in the presence and absence of drug. Filledsymbols show data for control alpha (circles, n = 4) and alpha in the presence of 50 nM(-)D888 (triangles, n = 5). Opensymbols show mean data (±S.E.) for control, Y1463A,A67S,I70A triple mutant (squares, n = 3), and YAI in the presence of 5 µM(-)D888 (inverted triangles, n = 4). Smoothlines represent fits of the mean data with relative current = 1/(1 + exp((V - V)/k)) the equation, and had the following values: alpha control, V = -17.7 mV, k = 5.3; 50 nM(-)D888 with alpha, V = -33.6 mV, k = 10.7; YAI control, V = -9.3 mV, k = 5.7; 5 µM D888 with YAI, V = -26.2 mV, k = 7.6. D, alpha-helical model of segment IVS6 of alpha. The proposed positions of residues in IVS6 are shown with Tyr-1463 facing the lumen.



In contrast to their lack of effect on channel activation, phenylalkylamines cause Ca channel inactivation curves to shift in the hyperpolarizing direction, indicating that block by these compounds is more potent at depolarized potentials where inactivation is favored(24, 25) . Although control inactivation curves for YAI are approximately 8 mV more positive than wt alpha (wt, V = -17.7 ± 2.9 mV; YAI, V = -9.3 ± 1.3 mV), both show an approximately 15-mV hyperpolarizing shift at a drug concentration equivalent to the IC (Fig. 4C). Since inactivation is minimal at the holding potential used in these experiments (-60 mV) and the drug-induced shift in V for inactivation is similar for mutant and wt, the decrease in(-)D888 potency cannot be ascribed to changes in the intrinsic voltage dependence of channel inactivation in the mutants.

Surprisingly, the apparent reversal potential of peak calcium channel currents in the mutant YAI was 15 mV more negative than in wt alpha (wt, E = 61.3 ± 4.4 mV, n = 10; YAI, E = 46.4 ± 1.8 mV, n = 5) (Fig. 4, A and B). This shift was apparent in all mutant channels in which Tyr-1463 was replaced by alanine or isoleucine (Y1463A, E = 47.6 ± 2.0 mV; YI, E = 47.2 ± 5.8 mV; YAI(N), E = 48.9 ± 2.0 mV) and the N-type channel in PC12 cells (E = 50.7 ± 1.1 mV) but not in other mutant channels (A1467S, E = 65.4 ± 7.4 mV; I1470A, E = 65.5 ± 1.6 mV; AI, E = 56.1 ± 3.7 mV). The change in E for Y1463A is not observed if the outward gradient of N-methyl-D-glucamine is abolished by substitution of 150 mMN-methyl-D-glucamine for Tris in the extracellular solution, indicating that this mutation allows permeation of N-methyl-D-glucamine. These results suggest that Tyr-1463 plays an important role in the selectivity of ion permeation as well as in high affinity binding of phenylalkylamines and therefore is likely to face the channel pore. The amino acid corresponding to Tyr-1463 in segment IVS6 of the brain type IIa Na channel, Ile-1760 (Fig. 3A), is also implicated in the ion conduction pathway since mutation of this amino acid allows a permanently charged local anesthetic to reach its receptor site from the extracellular side(14) .

A High Affinity Receptor Site for the Phenylalkylamine(-)D888 in the Intracellular Mouth of the Pore of L-type CaChannels

Our results implicate amino acid residues Tyr-1463, Ala-1467, and Ile-1470 in formation of the high affinity receptor site for phenylalkylamines and suggest that Tyr-1463 may contribute to the lining of the channel pore. Arranging alpha IVS6 residues in an alpha-helix (Fig. 4D) suggests that Tyr-1463, Ala-1467, and Ile-1470 are aligned on the same face of the helix. We propose that this face of the helix lines the intracellular end of the pore and forms part of the high affinity phenylalkylamine binding site in L-type calcium channels. Local anesthetics occupy a structurally similar receptor site in the pore of Na channels(14) . Thus, these two major classes of clinically important ion channel-blocking drugs may occupy analogous receptor sites formed in part by amino acid residues in transmembrane segments IVS6 of Na and Ca channels. These segments may also form part of the lining of the intracellular mouths of the pores of these structurally and functionally homologous proteins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant P01 HL44948 and by a research grant-in-aid from the American Heart Association (to W. A. C.), a postdoctoral research fellowship from the National Institutes of Health (to G. H. H.), and a postdoctoral research fellowship from the Muscular Dystrophy Association (to B. D. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: NGF, nerve growth factor; AI, A1467S,I70A; YA, Y1463A,A67S; YI, Y1463A,I70A; YAI, Y1463A, A67S,I70A; wt, wild type.


ACKNOWLEDGEMENTS

We thank Drs. K. Campbell, S. Ellis, M. Harpold, A. Schwartz, and T. Snutch for cDNA clones.


REFERENCES

  1. Snutch, T. P., and Reiner, P. B. (1992) Curr. Opin. Neurobiol. 2,247-253 [Medline] [Order article via Infotrieve]
  2. Catterall, W. A. (1994) Curr. Opin. Cell Biol. 6,607-615 [Medline] [Order article via Infotrieve]
  3. Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1987) Nature 328,313-318 [CrossRef][Medline] [Order article via Infotrieve]
  4. Hescheler, J., Pelzer, D., Trube, G., and Trautwein, W. (1982) Pflügers Arch. Eur. J. Physiol. 393,287-291
  5. Fleckenstein, A., and Fleckenstein-Grun, G. (1980) Eur. Heart J. 1,15-21 [Medline] [Order article via Infotrieve]
  6. Goll, A., Ferry, D. R., Striessnig, J., Schober, M., and Glossmann, H. (1984) FEBS Lett. 176,371-377 [CrossRef][Medline] [Order article via Infotrieve]
  7. Erdmann, R., and Luttgau, H. C. (1989) J. Physiol. (Lond.) 413,521-541 [Abstract]
  8. Striessnig, J., Knaus, H.-G., Grabner, M., Moosburger, K., Seitz, W., Lietz, H., and Glossmann, H. (1987) FEBS Lett. 212,247-253 [CrossRef][Medline] [Order article via Infotrieve]
  9. Striessnig, J., Glossmann, H., and Catterall, W. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,9108-9112 [Abstract]
  10. Lopez, G. A., Jan, Y. N., and Jan, L. Y. (1994) Nature 367,179-182 [CrossRef][Medline] [Order article via Infotrieve]
  11. Taglialatela, M., Champagne, M., Drewe, J. A., and Brown, A. M. (1994) J. Biol. Chem. 269,13867-13873 [Abstract/Free Full Text]
  12. Shieh, C., and Kirsch, G. E. (1994) Biophys. J. 67,2316-2325 [Abstract]
  13. Choi, K. L., Mossman, C., Aub'e, J., and Yellen, G. (1993) Neuron 10,533-541 [Medline] [Order article via Infotrieve]
  14. Ragsdale, D. S., McPhee, J. C., Scheuer, T., and Catterall, W. A. (1994) Science 265,1724-1728 [Medline] [Order article via Infotrieve]
  15. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,488-492 [Abstract]
  16. Snutch, T. P., Tomlinson, W. J., Leonard, J. P., and Gilbert, M. M. (1991) Neuron 7,45-57 [Medline] [Order article via Infotrieve]
  17. Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,2424-2428 [Abstract]
  18. Pragnell, M., Sakamoto, J., Jay, S. D., and Campbell, K. P. (1991) FEBS Lett. 291,253-258 [CrossRef][Medline] [Order article via Infotrieve]
  19. Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W. J., Hui, A., Schwartz, A., and Harpold, M. M. (1988) Science 241,1661-1664 [Medline] [Order article via Infotrieve]
  20. Margolskee, R. F., McHendry-Rinde, B., and Horn, R. (1994) BioTechniques 15,906-911
  21. Blaber, M., Zhang, K. J., and Matthews, B. W. (1993) Science 260,1637-1640 [Medline] [Order article via Infotrieve]
  22. Dubel, S. J., Starr, V. B., Hell, J., Ahlijanian, M. K., Enyeart, J. J., Catterall, W. A., and Snutch, T. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,5058-5062 [Abstract]
  23. Grantham, C. J., Main, M. J., and Cannell, M. B. (1994) Br. J. Pharmacol. 111,483-489 [Abstract]
  24. Ehara, T., and Kaufmann, R. (1978) J. Pharmacol. Exp. Ther. 207,49-55 [Abstract]
  25. McDonald, T. F., Pelzer, D., and Trautwein, W. (1984) J. Physiol. (Lond.) 352,217-241 [Abstract]

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