- Email: [email protected]
More than a Ca2+ channel?
Acknowledgements I thank Drs S. Numa, T. Claudio and F. Hucho forproviding manuscriptsprior to publication. Work from my laboratory is supported by NIH grant NS21229.
R. M. (1986) Annu. Rev. Neurosci. 9, 383-413 3 Claudio, T. in Frontiers of Molecular
Biology, Molecular Neurobiology Volume (Glover, D. M. and Hames, B. D., eds), IRL Press (in press) 4 Imoto, K. et al. (1988) Nature 335, 645-648 5 Leonard, R. J., Labarca, C. G., Charnet, P., Davidson, N. and Lester, H. A. (1988) Science 242, 1578-1581 6 Guy, H.R. and Hucho, F. (1987) Trends Neurosci. 10, 318-321 7 Imoto, K. et al. (1986) Nature 324, 670-674 8 Giraudat, J., Dennis, M., Heidmann, T., Chang, J-Y. and Changeux, J-P.
9
10 11 12 13
14
(1986) Proc. Natl Acad. Sci. USA 83, 2719-2723 Hucho, F., Oberthur, W. and Lottspeich, F. (1986) FEBS Lett. 205, 137-142 Dani, J. A. and Eisenman, G. (1987) J. Gen. PhysioL 89, 959-983 Dani, J. A. (1989) J. Neurosci. 9, 882-890 Toyoshima,-C:-and Unwin, N. (1988) Nature 336, 247-250 Hilgenfeld, R. and Hucho, F. (1988) in Transport through Membranes: Carriers Channels and Pumps (Fullrnan, A. et al., eds), pp. 359-367, D. Reidel Mishina, M. etal. (1986) Nature 321, 406-411
More thana Caz+ channel? Bruce Bean Department of Neurobiology, Harvard Medical School,220 LongwoodAvenue, Boston, MA 02115, USA.
128
he central role played by voltage-dependent Caz÷ chanT nels in processes such as synaptic transmission, smooth and cardiac muscle contraction, and secretion has made them interesting to a wide variety of biologists. So it was natural that after successfully cloning the acetylcholine receptor and Na ÷ channels, Shosaku Numa's group turned part of their considerable energies to the Ca2+ channel. The starting point for this effort was the purification and partial amino acid sequencing of a presumed Ca 2÷ channel protein from skeletal muscle, identified by its high-affinity binding site for dihydropyridine (DHP) drugs. This family of drugs, which includes nifedipine and nitrendipine, is valuable clinically for treating hypertension and angina; the drugs act by blocking Ca"z+ channels in vascular smooth muscle and thereby relaxing arterioles. High-affinity binding sites for the drugs, generally assumed to be Ca2+ channels, are found in smooth muscle, cardiac muscle and brain. However, the highest density of DHP binding sites is in skeletal muscle, specifically localized in the transverse tubules (t-tubules), a system of infolded surface membrane reaching into the interior of the muscle fiber. The DHP receptor from skeletal muscle is a complex consisting of four polypeptides of molecular masses 175 kDa, 170 kDa, 52 kDa and 32 kDa 1. The DHP binding site is located on the 170 kDa peptide (called the cq-subunit), and it is this peptide that was sequenced via molecular cloning by Tsutomo
Tanabe and his colleagues in Numa's laboratory2 (see also Ref. 3). (In fact, at the time the cloning effort began, the distinction between the 170 kDa and the 175 kDa peptides had not yet been realized, and Numa reported at a recent meeting that his group first cloned and sequenced the 175 kDa peptide. The sequence of this peptide has recently been reported by another group3; it has no homology with channel-forming peptides and its function is unknown.) The DHP-binding peptide has a high degree of homology with the several types of voltage-dependent Na + channel that have been cloned4-6. Like the Na + channel, the DHP receptor contains four internal repeats, each of which contains six probable membranespanning segments. One of the segments in each repeat is closely homologous to the so-called $4 segment in Na + channels, hypothesized to be the voltage-sensor of the Na ÷ channel, which is also found in the voltage-dependent K ÷ channel encoded by the shaker locus of Drosophila'. It therefore seems reasonable to think that the DHP-binding peptide could form a voltage-dependent Ca2+ channel. However, although the mRNA for a rat brain Na + channel and the shaker K ÷ channel have been shown to produce functional channels when injected into Xenopus oocytes8'9, this has so far not been shown for the DHP receptor (and one guesses it is not for lack of trying). It may be that Xenopus oocytes simply happen to be a poor expression system for the protein, perhaps lacking the machinery for
© 1989, ElsevierScience Publishers Ltd, (UK) 0166- 2236/89/$02.00
15 Unwin, N., Toyoshima, C. and Kubalek, E. (1988)J. Cell Biol. 107, 1123-1138 16 Schofield, P. R. et a/. (1987) Nature 328, 221-227 17 Grenningloh, G. et al. (1987) Nature 328, 215-220 18 Karlin, A., Kao, P. N. and Dipaola, M. (1986) Trends Pharmacol. Sci. 7, 304-308 19 Boulter, J. eta/. (1986) Nature 319, 368-374 20 Hermans-Borgmeyer, I. et al. (1986) EMBO J. 5, 1503-1508 21 Noda, M. et al. (1983) Nature 302, 528-532 22 Takai, T. et al. (1985) Nature 315, 761-764
proper post-translational processing. (At a minimum, proteolytic processing seems likely, because the amino acid sequence deduced from the cDNA has a predicted molecular mass of 212 kDa, larger than the 170 kDa DHP-binding peptide purified from muscle.) More interestingly, it may be that other subunits of the DHP-binding complex are needed to form a functional Ca2+ channel. Another intriguing possibility is that the DHP receptor does not primarily function as a Ca2÷ channel even when normally present in skeletal muscle. The possibility of another function for this protein was first raised by electrophysiological experiments aimed at understanding one of the central problems of muscle physiology, excitation-contraction coupling (E-C coupling). Somehow, depolarization of the t-tubule membrane by an action potential causes release of Ca 2+ from the internal stores of the sarcoplasmic reticulum; the mechanism of this coupling is a long-standing puzzle. An important clue came in 1973, when Martin Schneider and Knox Chandler, studying the electrical capacitance of skeletal muscle membranes, discovered that depolarization of the muscle fiber membrane (including the t-tubule system) produced movement of electrical charges within the membrane 1°. Because the voltagedependence of the intramembrane charge movement was similar to that of contraction, they hypothesized that the charge movement arises from molecular rearrangement of intramembrane molecules that act as voltage sensors controlling E-C coupling. Subsequent work has tended to support this interpretation. The identity of the TINS, Vol. 12, No. 4, 1989
molecules generating this membrane-bound charge movement remained mysterious until recently, when Eduardo Rios, Gustavo Brum and Enrico Stefani found that the DHP drug nifedipine could partially inhibit the charge movement and that this depression was accompanied by inhibition of Ca2+ release from the sarcoplasmic reticulum 11'12 (see also Ref. 13). They therefore suggested that the molecules generating the charge movement are the DHP receptors present at high density in the t-tubule membrane, and that these molecules might serve a dual role, acting both as voltage-dependent Ca2+ channels and as voltage sensors for E-C coupling. This idea was supported by a separate series of experiments on mice with muscular dysgenesis, a recessive mutation in which skeletal muscle E-C coupling is disrupted. Kurt Beam and his colleagues found that besides lacking E-C coupling, skeletal muscle from mutant animals had no DHPsensitive slow Ca2+ current, even though other types of nerve and muscle Ca2÷ channel were present in mutant animals TM. Biochemical studies by others showed that DHP binding sites were greatly reduced in the mutant muscle 15. These observations supported the idea that the same DHP-binding molecule might underlie both slow Ca2+ current and E-C coupling. An obvious possibility is that the two are not really separate functions, that current flow through the channels might be required to trigger contraction (as is believed for cardiac and smooth muscle), but there is evidence against this, since some blockers of Ca2+ current do not inhibit E-C coupling 16'17. Instead, the same depolarization-induced conformational change that opens the pore of the Ca2+ channel may also somehow control the very different Ca2+-release channels in the sarcoplasmic reticulum membrane. In a sense, the Ca2+ channel function of the DHP receptor may be the less important one, since contraction does not depend on Ca2+ influx and only about 5% of the DHP receptors form open Ca2+ channels (at any one time) upon depolarization TM. Exactly how the DHP receptors in the t-tubule membrane are coupled to Ca 2+release channels in the sarcoTINS, Vol. 12, No. 4, 1989
plasmic reticulum membrane remains to be determined. The two membranes are separated by a gap of about 150 A in the regions of closest approach, but the gap is spanned by regularly arranged electron dense structures termed feet 19. Recent ultrastructural evidence suggests that the feet are cytoplasmic extensions of the protein that forms the Ca 2+release channel in the sarcoplasmic reticulum membrane 2°-22. Moreover, the feet appear to be directly linked to particles in the t-tubule membrane that are probably DHP receptors 21, supporting the original hypothesis of Schneider and Chandler of a direct connection between the voltage sensors in the t-tubule membrane and Ca 2+release channels in the sarcoplasmic reticulum membrane. Thus it is possible to imagine that movement of the voltage-sensing portions of the DHP receptor would be communicated directly to the Ca2+-release channel of the sarcoplasmic reticulum. According to this hypothesis, both functions of the DHP receptor would be mediated by the same voltage-sensing mechanism in the molecule. The discovery from cloning that the DHP-binding protein has strong homology with the Na + channel, especially in the $4 segment hypothesized to be the voltage-sensing region, was consistent with the idea that the protein could serve a dual function, but obviously provided no new evidence that it actually does serve either function. Now, in a collaboration between the Numa and Beam laboratories, Tanabe and colleagues have provided more direct experimental evidence for a dual role of the DHP receptor 23. They injected a plasmid containing the cDNA encoding the skeletal muscle DHP receptor into cultured muscle cells from dysgenic mice and found that the injected plasmid restored both E-C coupling and slow Ca2+ current. Furthermore, testing fragments of genomic DNA with probes from the DHP receptor cDNA, they found that the structural gene for the DHP receptor in dysgenic mice differs from the normal gene in at least two regions, and that dysgenic muscle has greatly reduced levels of mRNA hybridizing with a probe from normal DHP cDNA. Another study has shown that dysgenic
muscle is selectively lacking in the DHP-binding ocl-subunit of the receptor complex, while having at least one of the other subunits 24. The simplest interpretation of the experiments of Tanabe et al. is that the injected cDNA results in the expression of the oq-subunit that is missing in dysgenic muscle and that this subunit plays a direct role in both E-C coupling and in forming a functional Ca2÷ channel. Another possibility is that the injected DNA acts in a less direct way, for example somehow stimulating the expression of an existing gene in the dysgenic muscle or facilitating the function of an existing protein. It will thus be important to check that injected dysgenic muscle produces mRNA and Ul-peptides that correspond to those encoded by the injected cDNA. Injection of modified cDNA prepared by mutagenesis can provide such a test. Assuming that the O¢l-SUbunit coded for by the cloned cDNA is involved directly in both E-C coupling and Cae+ channel function, many questions remain. Does the same copy of protein play both roles at the same time, with a depolarization-induced movement of amino acid residues simultaneously opening a CaZ+-selective pore and communicating with the sarcoplasmic reticulum? Or are the peptides subserving E-C coupling and Ca2+ channels separate and maybe even physically different, perhaps because of different posttranslational processing? Finally, what is the role of the other subunits in either function? The story so far is a good illustration of how the enormous advances made possible by molecular cloning are intertwined with concurrent efforts to understand the physiological role of the molecules being cloned, in this case a line of research that began with biophysicists measuring the capacitance of a muscle fiber. Physiologists can hope that this point will not be lost on those administrators who may believe that the advent of cloning has made other sorts of biology obsolete. The story also illustrates that those in the forefront of channel cloning are themselves well aware that the determination of an amino acid sequence is only a beginning in the understanding of how a protein works. 129
Selected references 1 Campbell, K. P., Leung, A. T. and Sharp, A. H. (1988) Trends Neurosd. 10, 425-430 2 Tanabe, T. et al. (1987) Nature 328, 313-318 3 Ellis, S. B. et al. (1988) Science 241, 1661-1664 4 Noda, M. et al. (1984) Nature 312, 121-127 5 Noda, M. eta/. (1986) Nature 320, 188-192 6 Salkoff, L. et al. (1987) Science 237, 744-749 7 Papazian, D.M., Schwarz, T.L., Tempel, B. L., Jan, Y. N. and Jan, L. Y. (1987) Science 237, 749-753 8 Noda, M. eta/. (1986) Nature 322, 826-828 .
.
.
.
.
' I'
e',
i ~~ ~, '~ x"il '~~ l ~'~" i l ~ I ~i~! ~'l ~ !~" ~ i ~~ii,~'~=H|" ~ ~ | "~'~ . . . . . .
.,.,,,
........................ 0..........
9 Timpe, L. C. etal. (1988) Nature331, 143-145 10 Schneider,M. F. and Chandler, W. K. (1973) Nature 242, 244-246 11 Rios, E., Brum, G. and Stefani, E. (1986) Biophys. J. 49, 13a 12 Rios, E. and Brum, G. (1987) Nature 325, 717-720 13 Lamb, G. D. and Walsh, T. (1987) J. Physiol. (London) 393, 595-617 14 Beam, K. G., Knudson, C. M. and Powell, J. A. (1986) Nature 320, 168-170 15 Pincon-Raymond, M., Rieger, F., Fosset, M. and Lazdunski, M. (1985) Dev. Biol. 112,458-466 16 Gonzalez-Serratos,H., Valle-Aguilera, R., Lathrop, D. A. and del Carmen Garcia, M. (1982) Nature 298, 292-294
perspectives
17 McCleskey, E. W. (1985)J. PhysioL (London) 361,231-249 18 Schwartz, L. M., McCleskey, E. W. and Almers, W. (1985) Nature 314, 747-751 19 Franzini-Armstrong, C. (1970)J. Cell Biol. 47,488-499 20 Inui, M., Saito, A. and Fleischer, S. (1987) J. BioL Chem. 262, 1740-1747 21 Block, B. A., Imagawa, T., Campbell, K.P. and Franzini-Armstrong, C. (1988) J. Cell BioL 107, 2587-2600 22 Fill, M. and Coronado, R. (1988) Trends Neurosci. 1O, 453-457 23 Tanabe, T., Beam, K. G., Powell, J. A. and Numa, S. (1988) Nature 336, 134-139 24 Knudson, C. M. et al. (1989) J. Biol. Chem. 264, 1345-1348
on disease
Molecular genetic studies in the neuropsychiatric disorders Joseph B. Martin JosephB. Martin is Chiefof the NeurologyService andJulieanneDora Professorof Neurologyat Harvard MedicalSchool, Massachusetts GeneralHospital, FruitStreet, Boston, MA 02114, USA.
130
Within the past decade intensive moleculargenetic research has been carried out in the attempt to pinpoint chromosomal loci that may be linked to neuropsychiatric disorders. Joseph Martin reviews progress made in the applicabon of moleculargenetics to the study of diseases such as Huntington's disease, Alzheimer's disease, familial amyIoidotic polyneuropathy and inherited nervous system tumors.
Progress has continued since an earlier review ~ of the application of molecular genetics to the neuropsychiatric disorders. Designation of the chromosomal locus for several autosomal dominant disorders has been accomplished by use of markers displaying restriction fragment length polymorphisms (RFLPs) combined with linkage analysis (Table I and Fig. 1). The chromosomal loci for two autosomal recessive diseases, Friedreich's ataxia (chromosome 9ql .1-1.2) 2 and ataxia-telangiectasia (chromosome 11q22-23) 3 have also been identified, as has the protein abnormality of Duchenne muscular dystrophy 4'5.
marker has yet been found. Early speculations that genes found within the D4S43 or D4S95 region 8'9 might be candidates for HD have now been discounted by recognition of several genetic recombinations between the HD gene and these probes. Unfortunately, no cytogenetic clues to any abnormality in the 4p16.3 region in HD patients have been found. Efforts are currently underway to clone and sequence systematically the entire 4p16.3 region contained in cosmid or yeast artificial chromosome vectors. It is likely that between ten and 20 genes reside in this region. One possibility not yet excluded is that the HD gene resides in a fixed translocation from another chromosome that has been inherited on chromosome 4. The slow progress in identifying the HD gene emphasizes problems inherent to the linkage analysis approach, difficulties also experienced in defining the gene in cystic fibrosis.
Huntington's disease
Presymptomatic testing of HD
Huntington's disease (HD), an autosomal dominant disorder, was the first of the human genetic disorders to be assigned a chromosomal location by RFLPs and linkage analysis 6. Family studies using the G8 probe (locus D4S10), physically mapped to 4p16.3, have shown a positive linkage without any reported exception in more than 50 pedigrees worldwide, supporting the impression from clinical and epidemiological studies that Huntington's disease may have originated from a single mutation 7. Subsequent studies have revealed additional RFLPs on 4p16.3. Two new markers, C4H [locus D4S43 (Ref. 8)] and D4S95 (Ref. 9) are both closer to the HD gene than D4S10, but each is located proximal to the HD locus 1°. The HDGgene is estimated to lie not more than 1-1.5 x 10 bp from the telomere. To date, despite saturation of the 4p region with more than 100 chromosome-4-specific probes, none has been genetically or physically mapped to the other side of the HD locus, i.e. no flanking
The availability of DNA markers linked to the HD gene has made presymptomatic (predictive) testing for the disease possible. Such studies have now been undertaken at several centers (see Ref. 11 for our recent report). Our first tests used the marker D4S10, despite the calculated 4% recombination rate between this locus and the HD gene. We have now entered 55 participants into our program from which 28 test results were given, 26 for adults and two prenatal. Of these 28, eight were positive, ten were negative and ten were uninformative. We have found that use of the new probes improves results and increases the accuracy in informative pedigrees to about 98%. Small families limit the application of predictive testing in about 40% of cases. Our experience indicates that the presymptomatic test can be given safely with appropriate clinical precautions. Another recent report confirms our experience 12. No serious adverse effects have so far occurred.
© 1989.ElsevieSci r encePublishersLtd.(UK) 0166-2236/89/$02.00
TINS, VoI. 12, NO. 4, 1989
R. M. (1986) Annu. Rev. Neurosci. 9, 383-413 3 Claudio, T. in Frontiers of Molecular
Biology, Molecular Neurobiology Volume (Glover, D. M. and Hames, B. D., eds), IRL Press (in press) 4 Imoto, K. et al. (1988) Nature 335, 645-648 5 Leonard, R. J., Labarca, C. G., Charnet, P., Davidson, N. and Lester, H. A. (1988) Science 242, 1578-1581 6 Guy, H.R. and Hucho, F. (1987) Trends Neurosci. 10, 318-321 7 Imoto, K. et al. (1986) Nature 324, 670-674 8 Giraudat, J., Dennis, M., Heidmann, T., Chang, J-Y. and Changeux, J-P.
9
10 11 12 13
14
(1986) Proc. Natl Acad. Sci. USA 83, 2719-2723 Hucho, F., Oberthur, W. and Lottspeich, F. (1986) FEBS Lett. 205, 137-142 Dani, J. A. and Eisenman, G. (1987) J. Gen. PhysioL 89, 959-983 Dani, J. A. (1989) J. Neurosci. 9, 882-890 Toyoshima,-C:-and Unwin, N. (1988) Nature 336, 247-250 Hilgenfeld, R. and Hucho, F. (1988) in Transport through Membranes: Carriers Channels and Pumps (Fullrnan, A. et al., eds), pp. 359-367, D. Reidel Mishina, M. etal. (1986) Nature 321, 406-411
More thana Caz+ channel? Bruce Bean Department of Neurobiology, Harvard Medical School,220 LongwoodAvenue, Boston, MA 02115, USA.
128
he central role played by voltage-dependent Caz÷ chanT nels in processes such as synaptic transmission, smooth and cardiac muscle contraction, and secretion has made them interesting to a wide variety of biologists. So it was natural that after successfully cloning the acetylcholine receptor and Na ÷ channels, Shosaku Numa's group turned part of their considerable energies to the Ca2+ channel. The starting point for this effort was the purification and partial amino acid sequencing of a presumed Ca 2÷ channel protein from skeletal muscle, identified by its high-affinity binding site for dihydropyridine (DHP) drugs. This family of drugs, which includes nifedipine and nitrendipine, is valuable clinically for treating hypertension and angina; the drugs act by blocking Ca"z+ channels in vascular smooth muscle and thereby relaxing arterioles. High-affinity binding sites for the drugs, generally assumed to be Ca2+ channels, are found in smooth muscle, cardiac muscle and brain. However, the highest density of DHP binding sites is in skeletal muscle, specifically localized in the transverse tubules (t-tubules), a system of infolded surface membrane reaching into the interior of the muscle fiber. The DHP receptor from skeletal muscle is a complex consisting of four polypeptides of molecular masses 175 kDa, 170 kDa, 52 kDa and 32 kDa 1. The DHP binding site is located on the 170 kDa peptide (called the cq-subunit), and it is this peptide that was sequenced via molecular cloning by Tsutomo
Tanabe and his colleagues in Numa's laboratory2 (see also Ref. 3). (In fact, at the time the cloning effort began, the distinction between the 170 kDa and the 175 kDa peptides had not yet been realized, and Numa reported at a recent meeting that his group first cloned and sequenced the 175 kDa peptide. The sequence of this peptide has recently been reported by another group3; it has no homology with channel-forming peptides and its function is unknown.) The DHP-binding peptide has a high degree of homology with the several types of voltage-dependent Na + channel that have been cloned4-6. Like the Na + channel, the DHP receptor contains four internal repeats, each of which contains six probable membranespanning segments. One of the segments in each repeat is closely homologous to the so-called $4 segment in Na + channels, hypothesized to be the voltage-sensor of the Na ÷ channel, which is also found in the voltage-dependent K ÷ channel encoded by the shaker locus of Drosophila'. It therefore seems reasonable to think that the DHP-binding peptide could form a voltage-dependent Ca2+ channel. However, although the mRNA for a rat brain Na + channel and the shaker K ÷ channel have been shown to produce functional channels when injected into Xenopus oocytes8'9, this has so far not been shown for the DHP receptor (and one guesses it is not for lack of trying). It may be that Xenopus oocytes simply happen to be a poor expression system for the protein, perhaps lacking the machinery for
© 1989, ElsevierScience Publishers Ltd, (UK) 0166- 2236/89/$02.00
15 Unwin, N., Toyoshima, C. and Kubalek, E. (1988)J. Cell Biol. 107, 1123-1138 16 Schofield, P. R. et a/. (1987) Nature 328, 221-227 17 Grenningloh, G. et al. (1987) Nature 328, 215-220 18 Karlin, A., Kao, P. N. and Dipaola, M. (1986) Trends Pharmacol. Sci. 7, 304-308 19 Boulter, J. eta/. (1986) Nature 319, 368-374 20 Hermans-Borgmeyer, I. et al. (1986) EMBO J. 5, 1503-1508 21 Noda, M. et al. (1983) Nature 302, 528-532 22 Takai, T. et al. (1985) Nature 315, 761-764
proper post-translational processing. (At a minimum, proteolytic processing seems likely, because the amino acid sequence deduced from the cDNA has a predicted molecular mass of 212 kDa, larger than the 170 kDa DHP-binding peptide purified from muscle.) More interestingly, it may be that other subunits of the DHP-binding complex are needed to form a functional Ca2+ channel. Another intriguing possibility is that the DHP receptor does not primarily function as a Ca2÷ channel even when normally present in skeletal muscle. The possibility of another function for this protein was first raised by electrophysiological experiments aimed at understanding one of the central problems of muscle physiology, excitation-contraction coupling (E-C coupling). Somehow, depolarization of the t-tubule membrane by an action potential causes release of Ca 2+ from the internal stores of the sarcoplasmic reticulum; the mechanism of this coupling is a long-standing puzzle. An important clue came in 1973, when Martin Schneider and Knox Chandler, studying the electrical capacitance of skeletal muscle membranes, discovered that depolarization of the muscle fiber membrane (including the t-tubule system) produced movement of electrical charges within the membrane 1°. Because the voltagedependence of the intramembrane charge movement was similar to that of contraction, they hypothesized that the charge movement arises from molecular rearrangement of intramembrane molecules that act as voltage sensors controlling E-C coupling. Subsequent work has tended to support this interpretation. The identity of the TINS, Vol. 12, No. 4, 1989
molecules generating this membrane-bound charge movement remained mysterious until recently, when Eduardo Rios, Gustavo Brum and Enrico Stefani found that the DHP drug nifedipine could partially inhibit the charge movement and that this depression was accompanied by inhibition of Ca2+ release from the sarcoplasmic reticulum 11'12 (see also Ref. 13). They therefore suggested that the molecules generating the charge movement are the DHP receptors present at high density in the t-tubule membrane, and that these molecules might serve a dual role, acting both as voltage-dependent Ca2+ channels and as voltage sensors for E-C coupling. This idea was supported by a separate series of experiments on mice with muscular dysgenesis, a recessive mutation in which skeletal muscle E-C coupling is disrupted. Kurt Beam and his colleagues found that besides lacking E-C coupling, skeletal muscle from mutant animals had no DHPsensitive slow Ca2+ current, even though other types of nerve and muscle Ca2÷ channel were present in mutant animals TM. Biochemical studies by others showed that DHP binding sites were greatly reduced in the mutant muscle 15. These observations supported the idea that the same DHP-binding molecule might underlie both slow Ca2+ current and E-C coupling. An obvious possibility is that the two are not really separate functions, that current flow through the channels might be required to trigger contraction (as is believed for cardiac and smooth muscle), but there is evidence against this, since some blockers of Ca2+ current do not inhibit E-C coupling 16'17. Instead, the same depolarization-induced conformational change that opens the pore of the Ca2+ channel may also somehow control the very different Ca2+-release channels in the sarcoplasmic reticulum membrane. In a sense, the Ca2+ channel function of the DHP receptor may be the less important one, since contraction does not depend on Ca2+ influx and only about 5% of the DHP receptors form open Ca2+ channels (at any one time) upon depolarization TM. Exactly how the DHP receptors in the t-tubule membrane are coupled to Ca 2+release channels in the sarcoTINS, Vol. 12, No. 4, 1989
plasmic reticulum membrane remains to be determined. The two membranes are separated by a gap of about 150 A in the regions of closest approach, but the gap is spanned by regularly arranged electron dense structures termed feet 19. Recent ultrastructural evidence suggests that the feet are cytoplasmic extensions of the protein that forms the Ca 2+release channel in the sarcoplasmic reticulum membrane 2°-22. Moreover, the feet appear to be directly linked to particles in the t-tubule membrane that are probably DHP receptors 21, supporting the original hypothesis of Schneider and Chandler of a direct connection between the voltage sensors in the t-tubule membrane and Ca 2+release channels in the sarcoplasmic reticulum membrane. Thus it is possible to imagine that movement of the voltage-sensing portions of the DHP receptor would be communicated directly to the Ca2+-release channel of the sarcoplasmic reticulum. According to this hypothesis, both functions of the DHP receptor would be mediated by the same voltage-sensing mechanism in the molecule. The discovery from cloning that the DHP-binding protein has strong homology with the Na + channel, especially in the $4 segment hypothesized to be the voltage-sensing region, was consistent with the idea that the protein could serve a dual function, but obviously provided no new evidence that it actually does serve either function. Now, in a collaboration between the Numa and Beam laboratories, Tanabe and colleagues have provided more direct experimental evidence for a dual role of the DHP receptor 23. They injected a plasmid containing the cDNA encoding the skeletal muscle DHP receptor into cultured muscle cells from dysgenic mice and found that the injected plasmid restored both E-C coupling and slow Ca2+ current. Furthermore, testing fragments of genomic DNA with probes from the DHP receptor cDNA, they found that the structural gene for the DHP receptor in dysgenic mice differs from the normal gene in at least two regions, and that dysgenic muscle has greatly reduced levels of mRNA hybridizing with a probe from normal DHP cDNA. Another study has shown that dysgenic
muscle is selectively lacking in the DHP-binding ocl-subunit of the receptor complex, while having at least one of the other subunits 24. The simplest interpretation of the experiments of Tanabe et al. is that the injected cDNA results in the expression of the oq-subunit that is missing in dysgenic muscle and that this subunit plays a direct role in both E-C coupling and in forming a functional Ca2÷ channel. Another possibility is that the injected DNA acts in a less direct way, for example somehow stimulating the expression of an existing gene in the dysgenic muscle or facilitating the function of an existing protein. It will thus be important to check that injected dysgenic muscle produces mRNA and Ul-peptides that correspond to those encoded by the injected cDNA. Injection of modified cDNA prepared by mutagenesis can provide such a test. Assuming that the O¢l-SUbunit coded for by the cloned cDNA is involved directly in both E-C coupling and Cae+ channel function, many questions remain. Does the same copy of protein play both roles at the same time, with a depolarization-induced movement of amino acid residues simultaneously opening a CaZ+-selective pore and communicating with the sarcoplasmic reticulum? Or are the peptides subserving E-C coupling and Ca2+ channels separate and maybe even physically different, perhaps because of different posttranslational processing? Finally, what is the role of the other subunits in either function? The story so far is a good illustration of how the enormous advances made possible by molecular cloning are intertwined with concurrent efforts to understand the physiological role of the molecules being cloned, in this case a line of research that began with biophysicists measuring the capacitance of a muscle fiber. Physiologists can hope that this point will not be lost on those administrators who may believe that the advent of cloning has made other sorts of biology obsolete. The story also illustrates that those in the forefront of channel cloning are themselves well aware that the determination of an amino acid sequence is only a beginning in the understanding of how a protein works. 129
Selected references 1 Campbell, K. P., Leung, A. T. and Sharp, A. H. (1988) Trends Neurosd. 10, 425-430 2 Tanabe, T. et al. (1987) Nature 328, 313-318 3 Ellis, S. B. et al. (1988) Science 241, 1661-1664 4 Noda, M. et al. (1984) Nature 312, 121-127 5 Noda, M. eta/. (1986) Nature 320, 188-192 6 Salkoff, L. et al. (1987) Science 237, 744-749 7 Papazian, D.M., Schwarz, T.L., Tempel, B. L., Jan, Y. N. and Jan, L. Y. (1987) Science 237, 749-753 8 Noda, M. eta/. (1986) Nature 322, 826-828 .
.
.
.
.
' I'
e',
i ~~ ~, '~ x"il '~~ l ~'~" i l ~ I ~i~! ~'l ~ !~" ~ i ~~ii,~'~=H|" ~ ~ | "~'~ . . . . . .
.,.,,,
........................ 0..........
9 Timpe, L. C. etal. (1988) Nature331, 143-145 10 Schneider,M. F. and Chandler, W. K. (1973) Nature 242, 244-246 11 Rios, E., Brum, G. and Stefani, E. (1986) Biophys. J. 49, 13a 12 Rios, E. and Brum, G. (1987) Nature 325, 717-720 13 Lamb, G. D. and Walsh, T. (1987) J. Physiol. (London) 393, 595-617 14 Beam, K. G., Knudson, C. M. and Powell, J. A. (1986) Nature 320, 168-170 15 Pincon-Raymond, M., Rieger, F., Fosset, M. and Lazdunski, M. (1985) Dev. Biol. 112,458-466 16 Gonzalez-Serratos,H., Valle-Aguilera, R., Lathrop, D. A. and del Carmen Garcia, M. (1982) Nature 298, 292-294
perspectives
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on disease
Molecular genetic studies in the neuropsychiatric disorders Joseph B. Martin JosephB. Martin is Chiefof the NeurologyService andJulieanneDora Professorof Neurologyat Harvard MedicalSchool, Massachusetts GeneralHospital, FruitStreet, Boston, MA 02114, USA.
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Within the past decade intensive moleculargenetic research has been carried out in the attempt to pinpoint chromosomal loci that may be linked to neuropsychiatric disorders. Joseph Martin reviews progress made in the applicabon of moleculargenetics to the study of diseases such as Huntington's disease, Alzheimer's disease, familial amyIoidotic polyneuropathy and inherited nervous system tumors.
Progress has continued since an earlier review ~ of the application of molecular genetics to the neuropsychiatric disorders. Designation of the chromosomal locus for several autosomal dominant disorders has been accomplished by use of markers displaying restriction fragment length polymorphisms (RFLPs) combined with linkage analysis (Table I and Fig. 1). The chromosomal loci for two autosomal recessive diseases, Friedreich's ataxia (chromosome 9ql .1-1.2) 2 and ataxia-telangiectasia (chromosome 11q22-23) 3 have also been identified, as has the protein abnormality of Duchenne muscular dystrophy 4'5.
marker has yet been found. Early speculations that genes found within the D4S43 or D4S95 region 8'9 might be candidates for HD have now been discounted by recognition of several genetic recombinations between the HD gene and these probes. Unfortunately, no cytogenetic clues to any abnormality in the 4p16.3 region in HD patients have been found. Efforts are currently underway to clone and sequence systematically the entire 4p16.3 region contained in cosmid or yeast artificial chromosome vectors. It is likely that between ten and 20 genes reside in this region. One possibility not yet excluded is that the HD gene resides in a fixed translocation from another chromosome that has been inherited on chromosome 4. The slow progress in identifying the HD gene emphasizes problems inherent to the linkage analysis approach, difficulties also experienced in defining the gene in cystic fibrosis.
Huntington's disease
Presymptomatic testing of HD
Huntington's disease (HD), an autosomal dominant disorder, was the first of the human genetic disorders to be assigned a chromosomal location by RFLPs and linkage analysis 6. Family studies using the G8 probe (locus D4S10), physically mapped to 4p16.3, have shown a positive linkage without any reported exception in more than 50 pedigrees worldwide, supporting the impression from clinical and epidemiological studies that Huntington's disease may have originated from a single mutation 7. Subsequent studies have revealed additional RFLPs on 4p16.3. Two new markers, C4H [locus D4S43 (Ref. 8)] and D4S95 (Ref. 9) are both closer to the HD gene than D4S10, but each is located proximal to the HD locus 1°. The HDGgene is estimated to lie not more than 1-1.5 x 10 bp from the telomere. To date, despite saturation of the 4p region with more than 100 chromosome-4-specific probes, none has been genetically or physically mapped to the other side of the HD locus, i.e. no flanking
The availability of DNA markers linked to the HD gene has made presymptomatic (predictive) testing for the disease possible. Such studies have now been undertaken at several centers (see Ref. 11 for our recent report). Our first tests used the marker D4S10, despite the calculated 4% recombination rate between this locus and the HD gene. We have now entered 55 participants into our program from which 28 test results were given, 26 for adults and two prenatal. Of these 28, eight were positive, ten were negative and ten were uninformative. We have found that use of the new probes improves results and increases the accuracy in informative pedigrees to about 98%. Small families limit the application of predictive testing in about 40% of cases. Our experience indicates that the presymptomatic test can be given safely with appropriate clinical precautions. Another recent report confirms our experience 12. No serious adverse effects have so far occurred.
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TINS, VoI. 12, NO. 4, 1989