Molecular diversity of Ca2+ channel beta subunits - Semantic Scholar

Structure and Regulation of Cation Channels

Molecular diversity of Ca2+channel /?subunits Antonio Castellano* and Edward Perez-Reyest$ *Dpto de Fisiologia Medica y Biofisica, Universidad de Sevilla, 41009 Sevilla, Esparia and +Department of Physiology, Loyola University Medical Center, Maywood, Illinois 60 153, U.S.A

Introduction Voltage-gated Ca2+ channels have been extensively characterized in terms of their pharmacological, electrophysiological, biochemical, and molecular biological properties. Calcium channels are multisubunit complexes composed of an ion-conducting subunit, a ,, and smaller accessory subunits. Molecular cloning studies have concentrated on the a , subunit because it appears to determine the pharmacological and electrophysiological properties of the channel. In fact, expression of cloned a , subunits from skeletal muscle and cardiac muscle displayed I>-type CaZc-channel activity, whereas other distinct, yet related, a I subunits display N-type activity [l-31. So far, cDNAs from six a , genes have been cloned. Alignment of the deduced amino acid sequences reveals that these a,s are members of a superfamily that can be subdivided into I,-type and non-L-type Ca2+ channels. Despite experiments showing that the accessory subunits are capable of modulating the properties of a , subunits [2,4], cloning of these subunits has until recently received little attention. In this report, we summarize our cloning of Psubunit cDNAs derived from four distinct genes and present new data on alternatively spliced forms. We also summarize our expression studies using a representative member of each of these P subclasses.

Methods The methods used to screen rat brain cDNA libraries have been described previously [5-71. Specific hybridization was achieved by ensuring relatively high stringency during both the hybridization (43% v/v formamide, 37°C) and the final wash (0.2 x ssc,67°C). p Subunit cDNAs were amplified from poly(A)' RNA using the PCR on first-strand cDNA. The amplified fragments were subcloned and sequenced using methods described previously

[Sl. A full-length cDNA encoding the rabbit CaCh2a a , subtype was constructed in the plasmid pAGA-2 [8]. In vitro transcription and injection of

Abbreviation used: DHP, dihydropyridine. $To whom correspondence should be addressed.

oocytes was performed as described previously [7]. Currents were measured using a two-microelectrode voltage-clamp amplifier [7].

Results and discussion Cloning of /3 subunit isoforms

The cDNA encoding the skeletal muscle /?subunit was the first to be cloned [9]. The skeletal muscle dihydropyridine (DHP) receptor complex was purified, the P subunit was sequenced, and corresponding oligonucleotides were used to screen cDNA libraries. Northern blot analysis using the /3 cDNA as a probe indicated that the only other tissue that expressed a cross-reactive species was brain. But the brain mRNA was larger than that observed in skeletal muscle, raising the possibility that it was derived from a second P gene. Thus we decided to clone the cDNA that encoded this brain p. W e started by screening rat brain cDNA libraries using the rabbit skeletal muscle P-subunit cDNA as a probe. Two distinct classes of positive clones were detected. One class encoded a sequence that appeared to be a splice variant of the gene that encodes the skeletal muscle isoform. W e referred to this C-terminus splice variant as Plb[S]. The second class of clones gave much weaker signals, and encoded a similar, but novel, /3 subunit. The differences in the sequence led us to propose that this was encoded by a second gene, hence we named it B2 [ S ] . Northern blot analysis using p2 cDNA as probe indicated that mRNAs could be detected in brain, heart and lung. In order to prove that P2 was expressed in heart, we amplified it with PCR, and subcloned and sequenced it. The sequence from numerous clones demonstrated that it was indeed PL. The PI gene can be alternatively spliced in two regions: within the last exon, to give rise to the two carboxyl terminus variants noted above, and within an internal region. The genomic sequence encompassing this internal region has been cloned [lo]. One of these exons encodes 52 amino acids (PI;,),whereas the other encodes only seven (PI,,).In some cases, both of these exons can be skipped (Pld),but this leads to a shift in the reading frame and a stop codon soon thereafter (A. S. I,. Yu, A. Castellano, E. Perez-Reyes and J. Lytton, unpublished work). Another splice variant (PIC)has also been cloned from brain and heart [ 11-1 31.

483

Biochemical Society Transactions

Alignment of all known

484

/3 subunit isoforms

Asterisks denote amino acid residues that are conserved in all four /3 subtypes. Numbering at right indicates the relative amino acid number, which when emboldened indicates the final residue. The rabbit skeletal muscle PI, sequence is from [9], the rat brain PI, sequence from [ 191,the human brain PI, sequence from [ I I], the rat kidney sequences p,, and j34dfrom A. S. L. Yu, A. Castellano, E. Perez-Reyes and J. Lytton (unpublished work), the rat brain sequence from [S], the rabbit heart &,,sequence from [ 141, the rat brain &a sequence from [6],and the rat brain /34asequence from [7]. The rabbit heart &, rat brain &, and rat brain P3bare reported here for-the first time.

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This variant has the small internal exon found in Blbtogether with the short C-terminus found in Pla (see Figure 1). The functional significance of these splice variants is not known. T o determine whether the p2 gene is similarly spliced at the internal exon, we amplified this region using PCR with primers based on regions of conserved sequences. PCR was performed on brain and heart cDNA, then the product was subcloned and sequenced. As observed for B,, three splice variants of B2 were found. These splicing events

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occurred in the same region as /?,and had the same pattern. The exon used in BZaencodes 45 amino acids, which share sequence identity with /?,a, BZc has the same small sequence found in PI,, and PIC, and BZdis produced by skipping both exons, causing a reading frame shift and termination 26 amino acids later (Figure 1). All three variants have also been detected in rat kidney (A. S. I,. Yu, A. Castellano, E. Perez-Reyes and J. Lytton, unpublished work). A fourth B2 variant, BZb,has also been cloned from heart cDNA [ 141. The B2,, cDNA


is produced by alternative splicing of the first exon, leading to a novel N-terminal sequence. P2balso contains the large internal variant found in pza. In addition to p2 sequences, we also isolated novel cDNA sequences, which we called p3 and p4 [6,7]. These PCR-derived clones were used to screen rat brain cDNA libraries. Full-length clones were isolated and sequenced. Figure 1 shows an alignment of all the known /3 isoforms cloned before December 1993. Surprisingly, the rat brain p3 and p4are nearly identical to PICand PzCin the internal splice region. In addition, we found splice variants of p3 in rat brain and kidney, and of p4 in rat kidney. These variants were produced by skipping the small exon, and as noted from Pldand & this deletion causes a frame shift and termination (Figure 1). The functional activity of these truncated p s has not been determined. It is probable that they are simply the result of splicing errors. The tissue distribution of the p-subunit subtypes was determined by Northern analysis. Table 1 summarizes these results. Northern analysis demonstrates that the predominant species in heart is p2. More sensitive methods, such as PCR and cDNA library screening, demonstrate that PI, p 3 and p4are also expressed in the heart. However, determining where in the heart these p s are expressed will require further studies, such as in situ hybridization.

Functional expression of B-subunit isoforms

Our first studies concerning the functional roles of subunits used stable transfection of mammalian cells with the rabbit skeletal muscle L-type a , and PI subunits [4].The two main results from these studies were, first, that PI can modulate the bio-

physical properties of the CaZ+channel formed by a and second, that PI can stabilize the channel in a state that binds DHPs with high affinity. T o test the functional activity of p2,p3 and B4,we switched to the cardiac L-type a , and to the Xenopus laevk oocyte expression system. Injection of cRNA encoding a I alone leads to the appearance of large DHP-sensitive currents (150 nA in 40 mM Ra"). The activation kinetics can be fitted with two exponentials corresponding to a fast (3-6 ms t)and a much slower component (50-80 ms Z) [8]. Coinjection of a p subunit with the cardiac a , subunit causes many changes in the Ca'+-channel currents. In general, PI, p2, p 3 and p4 all caused qualitatively similar effects on a , , so only the differences will be noted. The most prominent effect of p s is to cause a 10-fold increase the amplitude of the Ca*+-channel currents. This level of stimulation appears to require adequate expression of /3 protein

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A second effect of p s is to shift the apparent activation threshold in the hyperpolarizing direction. This effect was revealed by plotting the stimulation induced by p, versus the test potential [8], which showed that the stimulation was greatest at threshold potentials. This shift was more pronounced in subsequent experiments, presumably as a result of more efficient expression. In general, all p s are capable of shifting the current-voltage relationship by 10 mV. This result has two important implications: p s can modulate the biophysical properties of a ,, rather than simply enhancing functional expression; and ps can modify one of the defining characteristics of the distinct Ca2+-channel types, their voltage-dependence. A third effect of Bs is to alter the kinetics of the Ca'+-channel current. As noted above, a l -

-

Tpbk l Tissue distribution of the P-subunit subtypes The Northern results are subjectively divided into strong and weak signals. In general, weak signals require > 24 h exposure of the blot of detection. These results are from this study and [5-7,9,12- 141

Northern blot

PCR or cDNA library

Strong

Weak

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Skeletal muscle, brain

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Skeletal muscle, brain heart Kidney Brain, heart, kidney Heart, lung, kidney, spleen, Brain, heart, kidney, skeletal muscle skeletal muscle Kidney Brain, heart, kidney

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induced currents have biphasic kinetics. /3s significantly accelerate these kinetics by both increasing the contribution of fast component and by accelerating the slow component. Caution must be taken in interpreting the results of these analyses because of possible errors inherent to clamping the whole oocyte membrane. For this reason, we have analysed the data using simple methods such as either time-to-half-peak or single exponentials [5-71. /3s also affect inactivation kinetics. The Ba2+ currents recorded from a ,-injected oocytes show very little decay during test pulses (Figure 2). In contrast, decay of the aI/3-induced currents was readily observable (Figure 2). The amount of decay was not current-dependent, indicating that Ba2 was not substituting for Ca2+ at a modulatory site [ 151. Measurement of steady-state inactivation is difficult because of both cumulative inactivation (rundown) and the overlap with activation. Using 5 s prepulses, a ,-induced currents inactivated less than 40%. The a,/3,-and a ,B,-induced currents inactivated to a greater extent (60-75%) over a similar voltage range [6,7]. B, and B2 (Figure 2) also affect inactivation of the cardiac a , , but to a lesser degree. These results demonstrate that /3s can accelerate activation and inactivation kinetics, as well as increasing the amount of inactivation +

observed. It should however be noted that considerable variability in these results was observed between oocyte batches. There are many problems in using Xenopus oocytes for expression of Ca2+ channels. One problem is that they contain endogenous Ca2+ channels. These channels are largely ignored because they are usually small ( < 2 0 nA). Unexpectedly, it was found that exogenous Ca2 -channel subunits could stimulate these endogenous currents [ 11,161. W e decided to exploit this interaction to study the effects of mammalian B s on a novel Ca2+-channel subtype. W e found that all B subtypes were capable of stimulating (three-fold) the amount of endogenous current, which is considerably less than that observed with the cardiac a,. The fold stimulation was similar at all test potentials, indicating no apparent effect on the voltage dependence of the current. The current-voltage relationship of the endogenous channels is shifted -20 mV with respect to I,-type a ,-induced currents, suggesting that they are more closely related to low-voltage activated Ca2+ channels. The expression of these endogenous Ca2+ channels also varies considerably between oocyte batches. Bs did not induce the appearance of currents in batches ( - 40%) with no detectable endogenous currents. Because we con+

Expression of /3s alters the kinetics of various Ca2+ channels ( a ) Representative traces from oocytes injected with the cardiac L-type a , alone , or with

either b2or b4.Test pulses were t o + 30 mV from a holding potential of - 60 mV. ( b ) Representative traces from either uninjected, or oocyte injected with &, or b4.Test pulses were to + 20 mV from a holding potential of - 80 mV. The charge carrier was 40 mM Ba2' [7]. The data were filtered at 200 Hz and digitized at loo0 Hz. Linear background and capacitive transient currents were subtracted using a P/4 prepulse routine. Residual capacitive current transients resulting from saturation of the data recording system are truncated. (0)

L -

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100 ms


firmed /!? expression in these batches using coexpression with the cardiac a,, this result supports the notion that /!? can not form an ion pore. /!?induced stimulation of endogenous channel activity correlated with the levels of endogenous current observed in uninjected oocytes. In two batches of oocytes, these /!?-stimulated currents had peak amplitudes of 500 nA. Thus important controls must include injections of /!? alone in every batch of oocytes. Another way to separate the contributions of endogenous and expressed channels would be to use a selective blocker of one of these currents, if available [ 17,181. W e exploited the amplifying effects of /!? to study the pharmacological properties of the endogenous channels. W e found that these channels are insensitive to I,-type Ca2+-channel modulators, such as the DHPs, nifedipine and Bay K8644 [7], and to FPI, 64176 (A. Castellano and E. Perez-Reyes, unpublished work). The endogenous channels could be blocked by the crude venom from the funnel web spider, Agelenopsis aperta. This block was investigated further in collaboration with the laboratory of Dr M. E. Adams (University of California, Riverside, CA, U.S.A.). These studies showed that both the polyamine and peptide fraction could block current. Further, this block could be mimicked by the purified peptide w-Aga-IIIa, but not w-Aga-IVa (J. D. Mills, A. Kondo, E. PerezReyes and M. E. Adams, unpublished work). In addition, /!?-stimulated endogenous currents could be inhibited by the snail toxin w-conotoxin-GVIA. These results demonstrate that oocytes can express channels that are similar to N-type CaZ+channels. Yet, the extent of w-conotoxin-GVIA block varied between batches, whereas the w-Aga-IIIa block was constant. This result may result from the expression of two channel types that are both blocked by w Aga-IIIa, while only one is blocked by o-conotoxinGVIA. Conclusive evidence for two channel types was provided by single-channel recording. Although the effects of the /!? subtypes on the cardiac a 1 were qualitatively similar, this was not the case with the endogenous oocyte channels. p,, p3 and /I4 stimulated peak currents with little effect on inactivation kinetics [6,7]. In contrast, P2 unexpectedly slowed inactivation of currents from endogenous oocyte Caz+ channels (Figure 2).Using batches that expressed large endogenous currents, Dr A. E. Lacerda (Baylor College of Medicine, Houston, TX, U.S.A.) was able to measure single channels from uninjected and P2- and B4-injected oocytes. Two significant findings resulted from this study (A. E. Lacerda, E. Perez-Reyes, X. Wei, A.

Castellano, L. Birnbaumer and A. M. Brown, unpublished work). First, he found two channel types that differed in their conductance (9 and 18 pS, respectively, with 115 mM Ba2+)and their apparent voltage sensitivity. Second, he found that both channels were modulated by the /!? subunits. P2 dramatically increased burst duration and the number of bursts occurring throughout the test pulse. The summed single-channel currents were very similar to those obtained with the whole cell clamp. In combination with the pharmacological studies, we conclude that oocytes express a T-type channel and an N-type channel. The ability of /!?sto interact with multiple channel types implies that there are conserved domains of interaction.

Conclusion Our studies have demonstrated that molecular diversity of Ca2+ channels is in part caused by the expression of at least four distinct /!? genes and alternative splicing of these genes. W e have also demonstrated that functional diversity of Ca2+ channels can be caused by the expression of these /!? isoforms together with distinct a , subunits. W e would like to acknowledge the help and guidance of Drs X. Wei, A. E. Lacerda, L. Cribbs, J. Lytton, J. D. Mills, M. E. Adams, A. M. Brown and L. Birnbaumer. This work was supported by American Heart Association Texas Affiliate Grant # 91G-171 and National Institute of Health Grant HL-46702.

S.,Lacerda, A. E., Horne, W., Wei, X. Y., Rampe, D., Campbell, K. P., Brown, A. M. and Birnbaumer, I,. (1989) Nature (London) 340,233-236 Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S. and Numa, S. (1989) Nature (London) 340,230-233 Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A,, McCue, A.F., Velicelebi, G., Ellis, S. B. and Harpold, M. M. (1992) Science 257, 389-395 Lacerda, A. E., Kim, H. S., Ruth, P., Perez-Reyes, E., Flockerzi, V., Hofmann, F., Birnbaumer, L. and Brown, A. M. (1991) Nature (London) 352, 527-530 Perez-Reyes, E., Castellano, A., Kim, H. S., Bertrand, P., Baggstrom, E., Lacerda, A. E., Wei, X. and Birnbaumer, I,. (1992)J. Biol. Chem. 267,1792-1797 Castellano, A,, Wei, X., Birnbaumer, L. and PerezReyes, E. (1993) J. Biol. Chem. 268,3450-3455 Castellano, A,, Wei, X., Birnbaumer, I,. and PerezReyes, E. (1993) J. Biol. Chem. 268, 12359-12366 Wei, X. Y., Perez Reyes, E., Lacerda, A. E., Schuster, G., Brown, A. M. and Birnbaumer, L. (1991) J. Biol. Chem. 266,21943-21947

1 Perez-Reyes, E., Kim, H.

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9 Ruth, P., Kohrkasten, A., Riel, M., Bosse, E., Kegulla, S., Meyer, H. E., Flockerzi, V. and Hofmann, F. (1989) Science 245, 11 15-1 118 10 Powers, P. A,, Liu, S., Hogan, K. and Gregg, R. G. (1992)J. Riol. Chem. 267,22967-22972 11 Williams, M. E., Feldman, Lj. €I., McCue, A. F., Hrenner, R., Velicelebi. G., Ellis, S. H. and Harpold, M. M. (1992) Neuron 8 , 7 1 4 4 12 Chang, C. F. and Hosey, M. M. (1992) Hiophys. J. 61, A245 13 Collin, T., Wang, J.-J., Nargeot, J. and Schwartz, A. (1993) Circ. Res. 72, 1337-1344 14 Hullin, K., Singer-Lahat, D., Freichel, M., Hiel, M., Dascal, N., Hofmann, F. and Flockerzi, V. (1992) EMHO J. 11,885-890

15 Yue, Ll. T., Hackx, 1'. H. and Imredy, J. 1'. (1990) Science 250, 1735-1738 16 Singer, Lj.? Hiel, M., Lotan, I., Flockerzi, V., Hofmann, I;.and Llascal, N. (1991) Science 253,1553-1557 17 Lory, P., Rassendren, I;.A,, Kichard, S., Tiaho, F. and Nargeot,J. (1990)J. Physiol. 429,95-112 18 Bourinet, E., Fournier, F., Nargeot, J. and Charnet, 1'. (1992) FEBS Lett. 299, 5-9 19 Pragnell, M., Sakamoto, J., Jay? S. D. and Campbell, K. 1'. (1991) FEBS Lett. 291, 253-258

Received 20 December 1993

Involvement of cation channels in autoimmune disease Angela Vincent*+, Richard Barrett-jolley*, Paul Shillito", Ian Hart*, David Beeson", Calman MacLennan*, Mike Nicolle", Beth Lang*, Mark Roberts*, Hugh Willisont and John Newsom-Davis* "Neurosciences Group, Institute of Molecular Medicine,John Radcliffe Hospital, Oxford OX3 9DU, U.K.and tDepartment of Neurology, University of Glasgow, Glasgow G5 I 4TF, U.K.

Introduction

Bungurus multicinctus, ["'I] a-Hungarotoxin, to label

It is 20 years since the first demonstration that the muscle weakness in myasthenia gravis is caused by autoantibodies against the nicotinic acetylcholine receptor at the neuromuscular junction. Since then, three other conditions in which serum antibodies cause abnormalities of neuromuscular transmission have been defined, and for two of these, substantial evidence suggests that the target is an ion channel (see Figure 1 for a brief description of neuromuscular transmission). More detailed reviews covering the clinical and immunological aspects of these conditions [l-31, and describing the clinical and experimental findings in patients with genetic disorders of neuromuscular transmission [4],have been presented.

AChR extracted from human muscle or the rhabdomyosarcoma cell line TE67 1. T h e antibodies exhibit high affinity towards and are very specific for the human AChR [5], and are assumed to require 'T cell help' [6]. All of the human AChR genes have now been cloned, including the adult E form and a new isoform of the a gene [7] which contains a 25-aminoacid insertion between residues 58 and 59, termed P3A. Both forms of the a-subunit mRNA are expressed in TE671 cells [8], in human muscle [7,9], and in fetal muscle at various stages of development [9]. AChR-specific T cells can be cloned from peripheral blood or thymus of myasthenia gravis (MG) patients using recombinant a subunit, and some have been characterized in terms of the AChR peptide sequence that they recognize and their class I1 restriction [lo]. One T-cell clone recognizes only the a subunit that includes the extra sequence P3A; another T cell, from a healthy individual, recognizes only the a subunit that lacks this extra sequence [ 111. Interestingly, both clones can recognize purified muscle AChR, after it has been processed and presented by antigen-presenting cells, indicating that both isoforms of the AChR are present in adult human muscle [ 111. It is now possible to look for ways of inducing tolerance in or deleting these potentially pathogenic self-reactive T cells. Treatment of the fully charac-

Myasthenia gravis Anti-acetylcholine receptor (anti-AChR) antibodies are present in >85% of patients with generalized weakness. T h e antibodies are measured by immunoprecipitation, using a fraction of the venom of Abbreviations used: ACh, acetylcholine; AChR, ACh receptor; LEMS, Lambert-Eaton myasthenic syndrome; MEPP, motor end-plate potential; MFS, Miller-Fisher syndrome; MG, myasthenia gravis; NM, neuromyotonia; SCLC, small cell lung carcinoma; VGCC, voltage-gated calcium channel; VGKC, voltage-gated potassium channel; VGSC, voltage-gated sodium channel. +Towhom correspondence should be addressed.