1999 evolution and diversity of mammalian sodium channel genes.pdf

7/28/2019 1999 Evolution and Diversity of Mammalian Sodium Channel Genes.pdf

http://slidepdf.com/reader/full/1999-evolution-and-diversity-of-mammalian-sodium-channel-genespdf 1/9

a ) i n t he hum a n a nd m ous e genes (P l um m er et al., 1998),om pa r e d w i t h a n a v e ra g e v a l ue of 86% f or h u m a n a n d

mouse coding seq uences (Ma ka lowski et al., 1996). P hosphor-lat ion of residues w ithin t he cytoplasmic loops is known toa v e di st i nct effect s on t he kinet i cs of di ffer ent cha nnel sMurphy et al., 1996; Sm ith a nd G oldin, 1996; Frohnw ieser et l . , 1997).

HumanSCN6A and MouseScn7a Appear

to be Orthologs of a Single Gene

SC N 6 A a n d SC N 7 A wer e gi ven di ffer ent gene s y m b ol swhen they were mapped in human and mouse, respectively

George et al., 1994; Potts et al., 1993). How ever, the un usua lroperties t hey sha re a nd the la ck of evidence for t wo genes

within any one species suggest that SC N 6 A a nd SC N 7 A a r ec t ua l l y t he hum a n a nd m ous e or t hol ogs of a s i ngl e l ocus .

SC N 6 A i n hum a n a nd SC N 7 A in mouse ar e expressed in both

eur ona l a nd nonneur ona l t i s s ues , unl i ke t he ot her c ha n-els. Neither SC N 6 A nor SC N 7 A generates sodium currents

when the cDNAs are expressed in Xenopus oocytes (Felipe et l . , 1994; Akopia n et al ., 1997). The protein sequence of

SC N 6 A a n d SC N 7 A diverges from the other family membersby 50%, including cha nges in tw o critical d omains a ffectingvoltage sensitivity and ion selectivity (Gautron et al., 1992;George et al., 1992; Felipe et al., 1994; Akopian et al., 1997).The 68% amino acid identity of human SC N 6 A a nd m ous eSC N 7 A is consistent with the expecta tion for orthologousgenes (Felipe et al., 1994). However, this gene appears to bed iv er g in g m u ch m or e r a p id ly t h a n t h e ot h e r ␣ subunits.Human SCN8A and mouse SCN8A, for example, are 98.5%identical in a mino a cid sequence (Plummer et al., 1998).

Alternative Splicing of Neuronal SodiumChannels

Three si tes of a l ternat ive splicing furt her increase t he di-versity of neuronal sodium channel isoforms in mammaliantissues. These isoforms were first identified as cDNAs andlater explained by splicing mechanisms. The a lternat ive ex-ons 5N a nd 5A ar e separ at ed by less tha n 100 bp and encode

segments S 3 and S 4 of domain I in SC N 2 A, SC N 3 A, SC N 8 A,

a n d S C N 9 A ( S a r a o et a l . , 1991; G usta fson et a l . , 1993;Belcher et al ., 1995; Plummer et al ., 1997). Expr ession ofexon 5N pr edom i na t es i n t he neona t a l per i od, a nd exon A

FIG. 2. The ma mma lia n s odium cha nnel ␣ subunit genes are located in four paralogous chromosome regions. The ancestral chordate

enome is proposed to contain one copy of each gene listed a t the left . D uplicat ion events generated four para logous chromosome segments.

ndependent duplication, deletion, and translocation events subsequently altered the gene content of the paralogous segments and dividedhe region containing H O X A a nd SCN5A into three unlinked segments. The chromosomal locations of the corresponding mouse linkageroups are provided in Table 1. G enes are listed in alpha betical order because t heir physical order is not completely known. G ene symbols:

QP, a qua porin; C O L A , collagen ␣; C A C N B , calcium channel ␤; E R B B , a via n eryt hrobla s t ic leukemia vira l oncogene homolog; E V X ,ven-skipped homeobox; HOX, antennapedia -like homeobox; I T G A , integrin ␣; I T G B , integrin ␤; K R T , kera t in gene clust er ; N E U R O D ,

eurogenic differentia tion; N F E , nuclear factor erythroid; RAR, retinoic acid receptor; S C N A , sodium cha nnel ␣ subunit ; SLC4, solute carrieramily 4, anion exchanger; WNT, wingless -related. L inkage dat a from OMIM (Online Mendelian Inherita nce in Ma n, ht tp://ww w.ncbi.nlm.ih.gov/omim); TTX, tet rodotoxin; S , sensit ive, R, r esista nt .

325E V OL U TI O N O F TH E S O D I U M C H AN N E L G E N E F AM I L Y

redominat es in th e a dult . E xon 5N a nd exon 5A differ by aonsistent amino acid difference: the residue is uncharged inhe neona t a l i s ofor m a nd nega t i v el y c ha r ged i n t he a dul tsoform (Fig. 1, a rrow). The functiona l consequence of t hisonserved a mino acid substi tution is not known.

The alternative exons 18N and 18A of SC N 8 A encode the3 a nd S 4 s egm ent s of dom a i n I I I (P l um m er et al., 1997).

These exons share several features with exons 5A and 5N.oth pa irs of exons encode the S3 an d S4 segments of domain

II. Both pairs of exons are developmentally regulated withxon N expressed in ea rly d evelopment a nd exon A expressedn a dult . The genomic orga nizat ion of both pairs of exons isi m i l a r , wi t h t he ups t r ea m , neona t a l exon s epa r a t ed fr omhe adult exon by a small intron of 100 to 500 bp. Surpris-ngly, exon 18N of SC N 8 A contains a stop codon that wouldesult in synt hesis of a truncat ed tw o-domain protein (Plum-

m er et al., 1997). The fun ction of such a tr unca ted protein isnclear. Similar transcripts of SC N 1 A a nd SC N 8 A contain-

ng stop codons in d omain II I h ave been isolat ed from ast ro-ytes and neuroblastoma cells (Oh and Waxman, 1998).

Finally, alternative splicing in the first cytoplasmic loop ofSC N 1 A a nd SC N 8 A gener a t es t w o t r a ns c r ipt s t ha t di ffer b y10 amino a cids, due t o uti l ization of a l ternat ive splice donorsites in exon 10B (Schaller et al., 1992; Dietrich et al., 1998;Plummer et al ., 1998). In contra st to the neuron-specifi cgenes , no a l t er na t e t r a ns c r i pt s of SC N 4 A or SC N 5 A h a v ebeen identified.

Effects of Interaction with AuxiliarySubunits

Additiona l functiona l diversity of sodium cha nnels is gen-erated by interaction with the auxil iary subunits ␤1 a nd ␤2,encoded by the genes SC N 1 B a nd SC N 2 B (Ta ble 1). B oth ␤1a nd ␤2 cont a i n a s ingl e t r a ns m em br a ne s egm ent , a n i nt r a -cellular C-terminal domain, a nd a n extra cellular N-terminaldomain with an immunoglobulin-like (Ig) fold (Fig. 1), buttheir amino acid sequences are not related.

SC N 1 B is expressed in neurons, skeleta l muscle, a nd ca r-dia c muscle (Isom et al., 1992). Coexpression of SC N 1 B w i t h␣ s ubuni t s fr om b r a i n a nd s kel et a l m us cl e a cceler a t es t heki net i c s of c ha nnel a c t i v a t i on a nd i na c t i v a t i on, a l t er s t hevolta ge dependence of ina ctivat ion, an d increases peak cur-rent (Isom et al., 1992; Patton et al., 1994). Mut a tion of th e Igfold a bolished t he effect of SC N 1 B on ␣ subunit ina ctivat ion(McCormick et al., 1998; Wa lla ce et al., 1998). Coexpressionof SC N 1 B w i t h t h e c a r d ia c ␣ subunit SC N 5 A increases so-dium current but does not al ter channel kinetics (Qu et al.,

1995).SC N 2 B is expressed only in the nervous system (Wollner et

al ., 1987; Isom et al., 1995) and is covalently bound to the ␣

subunit by disulfide bonds (Messner and Catterall , 1985).SC N 2 B also conta ins a n extra cellular Ig fold w ith h omologyto the neural cell adhesion molecule contactin (Isom et al.,

1995) and may play a role in response to extracellular sig-nals. Coexpression of SC N 2 B alters the voltage dependenceand kinetics of inactivation of ␣ subunits and enhances lo-calizat ion in t he cell membra ne (Isom et al., 1995).

SodiumChannel Mutations and Inherited Disease

F our s odium c ha n nel ␣ s u b u n it s a n d o n e ␤ s u b u n it h a v eb ee n a s s oc ia t e d w i t h i n h er i t e d d i s ea s e i n h u m a n p a t i e n t sa n d m ous e m ut a nt s (Ta b l e 3 ). The s i t e-di r ect ed m ut a t i onof S CN 2 A, G AL8 79 -8 81 Q3, r es ul t s i n s l ow ed c ha nn el i n-a c t i v a t i o n ( K o n t i s a n d G o l d i n , 1 9 9 3 ) , a n d e x p r e s s i o n i n

TABLE 2

Conservation of Various Protein Domains i n So-dium C hannels from Four Paralogous ChromosomeSegments

SCN8A domain

%a mino a cid s equence ident it y t o

SCN 2A SCN 4A SCN 5A

D oma in I 79% 75% 69%C y t opla smic loop I /I I 51% L ow 42%

D oma in I I 93% 89% 80%C y t opla smic loop I I /I I I 65% 41% L ow D oma in I I I a nd I V 86% 84% 81%

Note. P e r ce n t a g e a m i n o a c id s eq u e nce i d en t i t y w a s ca l cu l a t edus ing t he B ESTFI T progra m in t he GCG pa cka ge. L ow , dif ferentnumbers of exons a nd high degree of divergence ma ke a lignmentu n r el ia b l e. G e n B a n k c it a t i o n s: h u m a n S C N 8 A , AF049617; r a t

Scn2a, X03639; human SCN4A, L04236; human SCN5A, M77235.

FIG. 3. P hylogenet ic rela t ions hip of ma mma lia n volt a ge-ga t edodium cha nnel ␣ subunit genes. Pr otein sequences from rodent a nd

Drosophila sodium channel genes were aligned using CLUSTAL W

ersion 1.6 softwa re (Thompson et al. , 1994). The aligned regiononta ins 1154 a mino a cids (approximately 2

3 of the total) correspond-ng to rat Scn1a residues 105–273, 329–423, 763–992, and 1215–874 (GenB an k Accession N o. X03638). The diver gent portions of t he

N-t ermina l region, C-t ermina l region, a nd cyt opla s mic loops t ha t

ould not be aligned were not included in the analysis. The alignedequences w ere a na lyzed w it h t he P R OTP A R S progra m (prot einequence pa rsimony meth od) from P HYL IP version 3.5 (ht tp://evo-ution.genet ics.wa shing ton.edu/phylip.ht ml). The numbers a t nodes

re boot s t ra p va lues for 100 replica t es of t he pa rs imony a na lys is .enBank accession numbers: rat Scn1a, X03638; rat Scn2a, X03639;

a t Scn3a, Y00766; r at Scn4a, M26643; ra t Scn5a, M27902; ra tcn7a, Y09164; mouse Scn8a, AF049617; ra t Scn9a U79568; rat

cn10a, U 53833; ra t Scn11a, AF059030; Drosophila P ar a, M32078;

Drosophila DSC1, X14394-8. The analysis was carried out with theodent s equences beca us e s equence is not a va ila ble for t he huma n

enes SCN1A, SCN3A, a nd SCN11A. The chromosoma l locationshown in the figure are for the human orthologs (Table 1), becauseeveral of the rat genes have not been mapped to chromosomes.

26 P L U M M E R A N D M E I S L E R

r a n s g e n ic m i ce r e s u l t s i n f oca l m ot o r a b n or m a l i t i es a n de iz u r e s ( K ea r n e y , J . A. , P l u m m er , N . W. , S m i t h , M . R . ,

K a pur , J . , G oldi n, A. L. , a nd M ei s l er , M . H . , m a nus c r i pt i nr ep a r a t i on ). H u m a n d i s ea s e m u t a t i on s w e r e fi r s t d i s co v-r ed i n S C N 4 A (P t a c ek et al ., 1991; Rojas et al ., 1991). Aa r ge s er i es of a l l el i c S C N 4 A m u t a t i on s h a v e b ee n i d e n t i -ed i n pa t i ent s wi t h m us c l e di s ea s e a nd func t i ona l l y c ha r -c t er i zed (Bul m a n, 1 9 9 7 ; Ca nnon, 1 9 9 7 ). Inher i t ed m ut a -ons i n S C N 5 A w e r e f o u n d i n p a t i e n t s w i t h t h e L o n g -Q T

y n d r o m e t y p e 3 , a d o m i n a n t l y i n h e r i t e d d i s e a s e c h a r a c -er i zed b y pr ol onged c a r di a c a c t i on pot ent i a l s (Bennet t et

l . , 1995; Wang e t a l . , 1 9 9 5 ). S pont a neous a l l el i c m ut a -

i o n s o f t h e m o u s e S C N 8 A c h a n n e l r e s u l t i n a v a r i e t y o feur ol ogi c a l a b nor m a l i t i es i nc l udi ng c hr oni c a t a xi a , dy s -

o n ia , a n d l et h a l p a r a l y s i s ( Me is l er et al ., 1997).M os t of t he m ut a t i ons i n SC N 4 A a nd SC N 5 A result in

l owed cha nnel i na c t iv a t i on a nd exhi bi t dom i na nt i nher i -ance (Table 3). These can be considered “gain of function”

muta tions since the mut an t protein ha s functiona l propertieshat differ from those of the wildtype. At the cellular level ,he dom i na nt phenot y pe i n t hes e het er ozy got es ca n b e ex-l a i ned b y t he per s i s t ent c ur r ent gener a t ed b y t he m ut a nthannels even in the presence of normal channel proteins. Inont r a s t , t he m i s sens e m ut a t i on i n S CN 8 A i s r eces si velynherited, and heterozygotes are unaffected. Since this mu-

a t i on i ncr ea s es t he degr ee of depol a r i za t i on r eq uir ed forhannel opening, in heterozygous cells the wildtype channels

will continue to open in response to a ppropriate depolariza -on a nd ma inta in n ormal responsiveness. The recessive in-er i t a nc e of nul l m ut a t i ons i n S CN 8 A dem ons t r a t es t ha t0%of normal activity of this channel is sufficient for normalhysiological function.

The fir s t m ut a t i on i n a s odi um c ha nnel ␤ subunit gene,SC N 1 B, was recently identified in a human family with gen-

ralized epilepsy with febrile seizures plus (GEFSϩ), a formf childhood epilepsy (Wa llace et al ., 1998). I n functionalssa ys, the cysteine to glycine muta tion appears to inactivat ehe ␤ subunit , resulting in slowed ina ctivat ion of ␣ subunits.

Volta ge-gat ed sodium chan nels a re a lso of m edical signif-can ce as ta rgets for na tura l neurotoxins an d synthetic pha r-

macological compounds including anti-convulsant drugs, an-st het i cs , a nd neur opr ot ect i v e dr ugs t ha t a m el ior a t e t he

effec t s of s t r oke (Ta y l or a nd N a r a s i m ha n, 1 9 9 7 ; R a gs da l ea nd Avoli, 1998).

OverlappingPhenotypes of Mutations in Different

Neuronal Ion Channels

The recent identification of disease mutations has revealedoverlapping phenotypes resulting from mutations in differ-ent voltage-gat ed chann els. E pilepsy a nd seizures ha ve beenassociated with mutations in six genes: a sodium channel ␣

subunit , a sodium channel ␤ subunit , a potassium channel ␣subunit , and the calcium chann el ␣, ␤, a n d ␥ subunits (Table

4 ). Inher i t ed a t a xi a ha s b een a s s oc i a t ed wi t h m ut a t i ons i nfour genes: a sodium channel ␣ s ub uni t a nd t hr ee c a l ci umchannel subunits. Mice with mutations in the ␣ subunits ofc a l c i um a nd s odi um c ha nnel s exhi b i t a s i m i l a r a t a xi c ga i t(Fletcher et al ., 1996; Kohrman et al ., 1996). The clinicalsimilari t ies of muta tions in th ese cha nnels is not surprising,in view of their coordinated roles in the generation of actionpotentia ls. The phenotypic overlap presents a clinical chal-l en g e f o r m u t a t i on i de nt i fi c a t ion i n f a m il ie s t h a t a r e t oos m a l l for l inka ge a na l y s is . In pedi gr ees l inked t o chr omo-some 2q24, for example, i t may be necessary to screen formutations in al l of the neuronal genes in this cluster.

Evolution of Tetrodotoxin ResistanceMost voltage-gat ed sodium chan nels a re blocked by tetro-

dotoxin, a neurotoxin present in pufferfish of the fa mily Te-tra odontida e. Among th e 11 cha nnels in Table 1, the cardiacchannel SC N 5 A a n d t h e n e u r o n a l c h a n n e l s SCN10A a ndS C N 1 1 A a r e u n iq u e i n t h e ir r e si st a n c e t o t e t r od ot ox in(Gellens et al., 1992; Akopia n et al., 1996; Sangameswaran et

al ., 1996; Ta te et al., 1998). These resistant genes contain apolar amino acid, cysteine or serine, at a position in the poresegment of doma in I t ha t is occupied by a n a romatic residuein the tetrodotoxin-sensitive genes. This aromatic residue ist hought t o i nt er a c t wi t h t he hy dr ophob ic s ur fa c e of t he t e-t r o d ot o xi n m o le cu l e (F o zz a r d a n d L i p ki n d , 1 996 ). S i t e -

directed muta genesis has confi rmed th e role of th is residue int he t et r odot oxi n r esi s t a nc e of SC N 5 A (Heinemann et al .,

1992b). Tetrodotoxin resistance can be generated experimen-tally by site-directed mutation of other residues that interact

TABLE 3

Overlapping Neurological Abnormalities Associated with Mutations in Voltage-Gated I on Channels

ha n nel G en e

P henot ype

At a xia D yst onia a P a ra ly sis Migra ine S eizures

Na ϩ SCN 8A m ed jo m ed J med,med tg —

SCN2Ab

noninactivat ing transgene a

SCN1B G E F SϩC a 2ϩ CACN A1A t ot t er in g F H M tottering

SCA6CACN B 4 l et h ar gi c l et h ar gi c

CACN G2 st ar gazer st ar gazer

CACN ␣2 ␦2 K ϩ K CN A1 t ar get ed n u l l , E AM

Note. G E F Sϩ, generalized epilepsy with febrile seizures plus; EA2, episodic ataxia type 2; SCA6, spinocerebellar ataxia type 6; FHM,amilial hemiplegic migraine. Uppercase, human gene symbol; lowercase italics, mouse mutant.

a Sprunger et al. (1998).b Kearney, J . A., Plummer, N. W., Smith, M. R., Kapur, J . , Goldin, A. L. , and Meisler, M. H., manuscript in preparation.

wi t h t he t oxi n (K ont i s a nd G ol di n, 1 9 9 3 ; F ozza r d a nd Li p-ind, 1996), but mutations at these si tes have not been ob-

erved in nature.

Origin of Muscle-Specific Sodium Channels

In contrast to the sodium-dependent action potentials iner t eb r a t e m us cl e, a c t i on pot ent i a l s i n m us cl e of i nver t e-rates such as crustaceans and insects are calcium-depen-ent (Fatt and Ginsborg, 1958; Suzuki and Kano, 1977). In

most invertebrates, the expression of sodium channels ap-ears to be l imited to neurons (Tseng-Crank et al ., 1991;

Oka m ur a et al ., 1994; Dyer et al ., 1997). Sodium currentsa ve been detected in muscle of a mphioxus, an invertebra tehor da t e wi t h t he pr edupli ca t i on chor da t e genom e (Ha gi -

wa r a a nd K i dokor o, 1971; Holl a nd et al ., 1994). The datauggest that expression in muscle was a characteristic of ann cestra l chordat e sodium channel. Tha t gene ma y ha ve hadua l expression in n eurons a nd muscle. Alternat ively, mus-l e s peci fic it y m a y ha v e a r i s en i ndependent l y i n t he m a m -

malian genes on chromosome 3 and chromosome 17. Furtherna l y si s of t he s eq uence a nd expr es s ion of t he a m phi oxusenes might shed l ight on t he evolution of muscle-specifi codium channels.

Future Prospects

The evolutiona ry r ela tionships am ong the ma mma lia n so-ium channel ␣ genes in chromosomal location, exon organi-ation, and tetrodotoxin sensitivity described here provide ar a m ewor k for under s t a ndi ng t he or i gi n a nd di v er genc e ofhi s gene fa m i l y . Com pa r i s on of t he pr om ot er r egions of

members of this gene family that differ in t issue specificityould contribute to understanding the molecular evolution ofegulatory elements. Specific functional roles of the cytoplas-

m i c l oop dom a i ns i s a not her i m por t a nt a r ea for fut ur e r e-earch.

The un ique physiological role of individual sodium chan nelenes remains one of the most interesting issues in channeliology. The mammalian genome contains five neuron-spe-ific sodium channels with highly conserved amino acid se-uences and overlapping expression patterns in the central

nd peripheral nervous system (Felts et al ., 1997). Differ-nces in electrophysiologica l properties (Ram a n et al., 1997;m i t h et al., 1998), subcellular localization (Westenbroek et l . , 1989; Toledo-Aral et al., 1997), and level of expression in

specific classes of neurons (Garcia et al., 1998) cont ribute tot hei r uni q ue func t i ons . D ur i ng t he pa s t dec a de, ext ens i v e

site-directed mutagenesis produced a solid understanding ofthe sh ar ed functiona l domains involved in channel functionstha t could be a ssayed in t he oocyte system. During t he com-ing decade, analysis of neurophysiological effects of humanand mouse mutations, observed i n v i v o, wi l l s hed l i ght onuni q ue dom a i ns a nd t he evol ut ion of funct i ona l di ver si t ywithin the sodium channel gene family.

ACKNOWLEDGMENTS

P r e pa r a t i on of t h i s r e v i ew w a s s u pp or t e d b y N I H G r a n t sNS034509 an d G M24872. We th ank P riscilla Tucker for assista ncew it h int erpret a t ion of t he phylogeny. We a re gra t eful t o our col-

leagues in the Meisler laboratory and at the University of Michiganfor ma ny st imulat ing discussions. An a nonmyous reviewer providedvaluable suggestions for improving the manuscript.

REFERENCES

Akopian, A. N., S ivilotti, L., a nd Wood, J . N. (1996). A tet rodotoxin-

resistan t volta ge-gat ed sodium channel expressed by sensory n eu-rons. N a t u r e 379: 257–262.

Akopian, A. N., Souslova, V., Sivilotti, L. , an d Wood, J . N. (1997).

Structure and distribution of a broadly expressed atypical sodiumchannel. F EBS Let t . 400: 183–187.

Anderson, P. A., Holman, M. A., and Greenberg, R. M. (1993). De-

duced amino a cid sequence of a putat ive sodium chan nel from thescyphozoan jellyfi sh Cyanea capil lata. Proc. Natl. Acad. Sci. USA

90: 7419–7423.

B a iley, W. J . , Kim, J . , Wa gner, G . P . , a nd R uddle, F. H. (1997).P hylogenetic reconstruction of vertebra te H ox cluster duplications.

M ol. Bi ol . Evol. 14: 843–853.

Belcher, S. M., Zerillo, C. A., Levenson, R., Ritchie, J . M., and Howe,J . R . (1995). Cloning of a s odium cha nnel a lpha s ubunit fromrabbit Schwann cells. Proc. Natl. Acad. Sci. USA 92: 11034–11038.

Benn ett , P . B. , Yaza wa , K., Makita , N., and G eorge, A. L. , J r . (1995).Molecular mechanism for an inherited cardiac arrhythmia. N a t u r e

376: 683–685.

B ulma n, D. E. (1997). P henot ype va ria t ion a nd new comers in ionchannel disorders. Hum. Mol. Genet. 6: 1679–1685.

B urges s , D. L . , Kohrma n, D. C. , Ga lt , J . , P lummer, N. W. , J ones ,J . M . , Spea r , B . , a nd M eis ler , M . H . (1995). M ut a t ion of a new sodium channel gene, Scn8a, in the mouse muta nt “motor endplat edisease.” Nat. Genet. 10: 461–465.

TABLE 4

The Mode of Inheritance of Sodium Channel Mutations Depends on the Alteration in Channel Function

G ene Tissue Mut a t ion E ffect on funct ion I nher it a nce P h en ot y pe

CN2A B ra in G AL879-881Q3 D ela yed ina ct iva tion,persistent current

D om in a n t F oca l m ot or a b nor m a li ti es ,generalized seizures

C N4A Muscle M1592V D ela y ed ina ct iva t ion,

persistent current

D om in a nt H YP P , episod ic m us cle

weakness, elevatedserum potassium

C N5A H ea r t ⌬K P Q 1505-1507 D e la y e d i na c t iv a t ion ,persistent current

D om in an t F at a l ca rd ia c a rr hy th mia

C N8A B r a in A1071T D epola rizin g sh ift in volt a gedependence of a ctivation

R ecessive At axia

C N8A B r a in Null C om plet e loss of a ct ivit y Recessive L et h a l pa r a lysis

Note. Representa tive examples of the known m uta tions in S CN2A (Kear ney, J . A., Plum mer, N. W., Smit h, M. R., Ka pur, J ., G oldin, A. L. ,n d Meisler, M. H., m an uscript in prepara tion), S CN4A (Ca nnon, 1997), SC N5A (B ennett et al., 1995), a nd SC N8A (Kohrma n et al., 1996;

Meisler et al., 1997).

u rglin, T. R., an d Ruvkun, G . (1993). The Caenorhabdi t is elegans

homeobox gene cluster. Cur r. Opin. Genet. D ev. 3: 615–620.

a nnon, S. C. (1997). From mut at ion to myotonia in sodium chann el

disorders. Neuromusc. Di sord ers 7: 241–249.

a n n o n , S . C . , H a y w a r d , L . J . , B e e ch , J . , a n d B r o w n , R . H . , J r .

(1 99 5). S o d iu m c h a n n e l i n a c t iv a t i o n i s i m p a i r ed i n e q u i n e

h y p e r k a l e m i c p e r i o d i c p a r a l y s i s . J . N eu r o p h y si o l . 73: 1892–

ib-Ha jj, S . D ., Tyrrell, L. , B lack, J . A., and Waxma n, S . G . (1998).NaN , a novel voltage-gat ed Na channel, is expressed preferentially

in peripheral sensory neurons and down-regulated after axotomy.

Proc. Natl. Acad. Sci. USA 95: 8963– 8968.

iet r ich, P . S. , M cG ivern, J . G. , Delga do, S. G. , Koch, B . D. , Eglen,

R . M . , H u n t e r , J . C . , a n d S a n g a m e sw a r a n , L . (1 99 8). F u n c-

t iona l a na lys is of a volt a ge-ga t ed s odium cha nnel a nd i t s s plice

v a r i a n t f r o m r a t d o r s a l r o o t g a n g l i a . J. Neurochem. 70: 2262–

yer , J . R . , J ohnst on, W. L . , Ca s t ellucci, V. F. , a nd Dunn, R . J .

(1997). Cloning and tissue distribution of the Aplysia Na ϩ cha nnel

alpha -subunit cDNA. DNA Cel l B iol . 16: 347–356.

s c a y g , A. , J o n es , J . M . , K e a r n e y , J . A. , H i t c h co ck , P . F . , a n d

M eis ler , M . H. (1998). C a lcium cha nn el ␤4 ( C AC N B 4 ): H u m a n

o r t h o l o g o f t h e m o u s e e p i l e p s y g e n e l e t h a r g i c . Genomics 50:14–22.

att , P. , and Ginsborg, B. L. (1958). The ionic requirements for the

p r od u ct i on of a c t i on p ot e n t i a l s i n c r us t a c ea n m u s cl e fi b e rs .

J. Physiol. 142: 516–543.

elipe, A., Knit tle, T. J ., D oyle, K. L. , a nd Tam kun, M. M. (1994).

P rima ry str ucture and d ifferentia l expression during development

a nd pregna ncy of a novel volt a ge-ga t ed s odium cha nnel in t he

mouse. J. Biol . Chem. 269: 30125–30131.

elts, P. A., Yokoyama, S. , Dib-Hajj, S. , Black, J . A., and Waxman,

S. G. (1997). Sodium channel ␣-subunit mRNAs I, II , III , NaG and

hNE (PN1): Different expression patterns in developing rat ner-

vous system. Br ain Res.Mol. Br ain Res. 45: 71–82.

letcher, C. F. , Lutz, C. M., O’Sullivan, T. N., Shaughnessy, J . D.,J r . , H a w kes, R . , Fra nkel, W. N. , Copela nd, N. G. , a nd J enkins ,

N. A. (1996). Absence epilepsy in tottering mutant mice is associ-

ated with calcium channel defects. Cell 87: 607–617.

ozzard, H. A., a nd Lipkind, G . (1996). The gua nidinium toxin bind-

ing site on the sodium channel. J p n . H ea r t J . 37: 683–692.

rohnw ies er , B . , Chen, L . Q. , Schreibma yer, W. , a nd Ka llen, R . G .

(1997). Modulation of the human cardiac sodium channel a-sub-

unit by cAMP-dependent protein kinase and the responsible se-

quence domain. J. Physiol. 498: 309–318.

a r c i a , K . D . , S p r u n g e r , L . K . , M e i s l e r , M . H . , a n d B e a m , K . G .

(1998). P ostnat al developmental increase in t he density of sodium

currents in murine motoneurons. J. N eur osci. 18: 5234–5239.

aut ron, S. , Dos Sant os, G ., Pinto-Henriq ue, D., Koulakoff, A., Gros,F., a nd B erwa ld-Netter, Y. (1992). The glia l voltage-gat ed sodium

channel: Cell- and tissue-specific mRNA expression. Proc. Nat l .

Acad. Sci. U SA 89: 7272–7276.

ellens, M. E ., George, A. L. , J r . , Chen, L. Q., Cha hine, M., Horn, R.,

B a r ch i , R . L . , a n d K a l l en , R . G . (19 92). P r i m a r y s t r u ct u r e a n d

functional expression of the human cardiac tetrodotoxin-insensi-

tive voltage-dependent sodium channel. Proc. Natl. Acad. Sci. USA

89: 554–558.

eorge, A. L. , J r . , Iyer, G . S. , Kleinfi eld, R., Kallen, R. G., an d B ar chi,

R. L. (1993). Genomic organization of the human skeletal muscle

sodium channel gene. Genomics 15: 598–606.

eorge, A. L., J r., Knittle, T. J ., and Tamkun, M. M. (1992). Molec-

ular cloning of an at ypical volta ge-gat ed sodium channel expressed

in huma n hea rt a nd ut erus : Evidence for a dis t inct gene fa mily.

Proc. Natl. Acad. Sci. USA 89: 4893– 4897.

eorge, A. L., J r., Kn ops, J . F., H an , J ., Finley, W. H., K nitt le, T. J .,

Tam kun, M. M., and B rown, G . B . (1994). Assignment of a huma n

volta ge-dependent sodium chan nel ␣-subunit gene (SCN 6A) to

2q21–q23. Genomics 19: 395–397.

Goldin, A . (1999). Diversit y of ma mma lia n volt a ge-ga t ed s odium

channels. I n “Molecular and Functional Diversity of Ion Channels

an d Receptors” (B. Rud y a nd P . Seeburg, Eds.), in press, New York

Academy of Sciences.

G ustafson, T. A., Clevinger, E. C ., O’Neill, T. J ., Yarowsky, P . J ., and

Krueger, B. K. (1993). Mutually exclusive exon splicing of type III

brain sodium channel alpha subunit RNA generates developmenta lly

regulated isoforms in rat brain. J. Biol. Chem. 268: 18648–18653.

Hagiwara, S., and Kidokoro, Y. (1971). Na and Ca components of action

potential in am phioxus muscle cells. J. Physiol. 219: 217–232.

Heineman n, S. H., Terlau, H ., St uhmer, W., Imoto, K., and Numa , S.

(1992a). Ca lcium channel cha racteristics conferred on the sodium

cha nnel by s ingle mut a t ions . N a t u r e 356: 441–443.

Heinemann, S. H., Terlau, H., and Imoto, K. (1992b). Molecular basis

for pharma cological differences between bra in a nd card iac sodium

channels. Pflu ger s A r ch . 422: 90–92.

Hille, B . (1992). “ I on C ha nnels of Excit a ble M embra nes , ” S ina uer

Associat es, Sun derland, MA.

Holla nd, P . W. H., G a rcia-Ferna n dez, J ., William s, N. A., and Sid ow,

A. (1994). Gene duplications and the origins of vertebrate devel-

opment. Development Suppl., 125–133.

Isom, L. L. , D e J ongh, K. S. , a nd Ca ttera ll, W. A. (1994). Auxiliary

subunits of voltage-gated ion channels. Neuron 12: 1183–1194.

I s om, L . L . , De J ongh, K. S. , P a t t on, D. E . , R eber, B . F. , Offord, J .,

Cha rbonnea u, H . , Wa ls h, K. , Goldin, A. L . , a nd Ca t t era ll , W. A .

(1992). Primary structure and functional expression of the beta 1

s ubunit of t he ra t bra in s odium cha nnel. Science 256: 839–842.

I s om, L . L . , R a gs da le, D. S . , De J ongh, K. S . , Wes t enbroek, R . E. ,

R eber, B . F. X. , S cheuer, T. , a n d C a t t era ll , W. A . (1995). S t ruc-

t ure a nd funct ion of t he ␤2 s ubunit of bra in s odium cha nnels , a

t r a n s m e m b r a n e g l y c o p r o t e i n w i t h a C A M m o t i f . Cell 83: 433–

J ones, J . M., Meisler, M. H., a nd Isom, L . L. (1996). Scn2b, a voltage-

gated sodium channel ␤2 gene on mouse chr omosome 9. Genomics 34: 258–259.

Kenyon, C . , a nd Wa n g, B . (1991). A clus t er of Ant ennapedia -class

h o m eo b ox g e n e s i n a n o n s eg m e n t e d a n i m a l . Science 253: 516–

Kohrma n, D. C ., Smith, M. R., G oldin, A. L. , Ha rris, J . , and Meisler,

M. H. (1996). A missense mutation in the sodium channel Scn8a is

res ponsible for cerebella r a t a xia in t he mous e mut a nt jo l t ing.

J . N eur osci. 16: 5993–5999.

Kontis, K . J . , and G oldin, A. L. (1993). S ite-directed mut agenesis of

t h e p ut a t i v e p o re r e gi on of t h e r a t I I A s od i um ch a n n e l. M o l .

Pharmacol. 43: 635–644.

Loughney, K., Kreber, R., and Ganetzky, B. (1989). Molecular anal-

ys is of t he para locus, a sodium channel gene in Dr osophila. Cell 58: 1143–1154.

Lundin, L. G . (1993). E volution of the vertebrat e genome as reflected

in paralogous chromosomal regions in man and the house mouse.

Genomics 16: 1–19.

Maka lowski, W., Zhang, J . , an d B oguski, M. S . (1996). Compar at ive

an alysis of 1196 orthologous mouse and huma n full-length m RNA

and protein sequences. Genome Res. 6: 846–857.

M a kit a , N. , Sloa n-B row n, K. , Weghuis , D. O. , R opers , H. H. , a nd

G eorge, A. L. , J r . (1994). G enomic orga nizat ion an d chromosomal

assignm ent of the huma n volta ge-gat ed Na(ϩ)cha nnel ␤-1 subu nit

gene (SCN1B ). Genomics 23: 628–634.

Marban, E. , Yamagishi, T., and Tomaselli, G. F. (1998). Structure

and function of voltage-gated sodium channels. J. Physiol . 508:647–657.

McClat chey, A. I., Cannon, S. C., Slaugenha upt, S. A., and G usella, J . F.

(1993). The cloning and expression of a sodium channel ␤1-subunit

cDNA from human brain. Hum. Mol. Genet. 2: 745–749.

McClatchey, A. I . , Lin, C. S . , Wan g, J ., Hoffman, E . P. , Rojas, C. , an d

Gusella, J . F. (1992). The genomic structure of the human skeletal

muscle sodium cha nnel gene. Hum. Mol. Genet. 1: 521–527.

McCormick, K. A., Isom, L. L. , Ra gsda le, D., Sm ith, D ., Scheuer, T.,

an d C at tera ll, W. A. (1998). Molecular d eterminan ts of Na ϩ cha n-

n e l f u n c t i o n i n t h e e x t r a c e l l u l a r d o m a i n o f t h e b e t a 1 s u b u n i t .

J. B iol . Chem. 273: 3954–3962.

M eis ler , M . H. , S prunger, L . K. , P lummer, N. W. , Es ca yg, A ., a nd

J ones, J . M. (1997). Ion channel muta tions in mouse models of

inherited neurological d isease. Ann. M ed. 29: 569–574.

Messner, D . J ., a nd C a tt era ll, W. A. (1985). The sodium cha nnel fr om

ra t bra in: Sepa ra t ion a nd cha ra ct eriza t ion of s ubunit s . J . B i ol .

Chem. 260: 10597–10604.

M urphy, B . J . , R ogers , J . , P erdichizzi , A . P . , Colvin, A . A. , a nd

Catterall, W. A. (1996). cAMP-dependent phosphorylation of two

s it es in t he a lpha s ubunit of t he ca rdia c s odium cha nnel. J . B i o l .

Chem. 271: 28837–28843.

Noda, M., Shimizu, S. , Tanabe, T., Taka i, T., Ka ya no, T., Ikeda , T.,

Ta ka ha s hi, H. , Na ka ya ma , H. , Ka noa ka , Y. , M ina mino, N. , Ka n-

g a w a , K . , M a t s u o , H . , R a f t e r y , M . A . , H i r o s e , T . , I n a y a m a , S . ,

Ha ya s hida , H. , M iya t a , T. , a nd Numa , S. (1984). P rima ry s t ruc-

t u r e o f El ectr ophoru s electr icus s odium cha nnel deduced from

cDNA sequence. N a t u r e 312: 121–127.h, Y. , a nd Wa xma n, S. G. (1998). Novel s plice va ria t ion of t he

sodium channel alpha subunit . NeuroReport 9: 1267–1272.

ka mura , Y. , Ono, F. , Oka ga ki, R . , Chong, J . A ., a nd M a ndel, G.

(1994). Neural expression of a sodium channel gene requires cell-

specifi c intera ctions. Neuron 13: 937–948.

at ton, D. E . , Isom, L. L. , Ca ttera ll, W. A., a nd G oldin, A. L. (1994).

The a dult bra in ␤1 s ubunit modifies a ct iva t ion a nd ina ct iva t ion

gat ing of multiple sodium chan nel ␣ subunits. J. Biol. Chem. 269:17649–17655.

lummer, N. W. , G a lt , J . , J ones , J . M . , B urgess , D . L . , S prunger,

L. K., Kohrman, D. C., and Meisler, M. H. (1998). Exon organiza-

tion, coding sequence, physical mapping, and polymorphic intra-

g e ni c m a r k e r s f o r t h e h u m a n n e u ro na l s od i u m c h a n n e l g e n e

SCN8A. Genomics. 54: 287–296.

lummer, N. W. , M cB urney, M . W. , a nd M eis ler , M . H. (1997).

Alterna tive splicing of the sodium chan nel SC N8A predicts a trun -

ca t ed t w o-doma in prot ein in fet a l bra in a nd non-neurona l cells.

J. B iol . Chem. 272: 24008–24015.

otts, J . F. , Regan, M. R., Rochelle, J . M., Seldin, M. F. , a nd Agnew,

W. S. (1993). A glial-specifi c volta ge-sensitive Na channel gene

ma ps close t o clus t ered genes for neurona l is oforms on m ous e

chromosome 2. Biochem. Biophys. Res. Commun. 30: 100–4.

tacek, L. J ., George, A. L., J r., Griggs, R. C., Tawil, R., Kallen, R. G.,

Barchi, R. L. , Robertson, M., and Leppert, M. F. (1991). Identifi-

ca t ion of a mut a t ion in t he gene ca us ing hyperka lemic periodic

pa ra lys is . Cell 67: 1021–1027.

u, Y., Isom, L. L., Westenbroek, R. E., Rogers, J . C., Tanada, T. N.,McCormick, K. A., Scheuer, T., and Catterall, W. A. (1995). Mod-

ula t ion of ca rdia c Na ϩ channel expression in Xenopus oocytes by

␤1 subunits. J. Biol . Chem. 270: 25696–25701.

agsd ale, D . S. , an d Avoli, M. (1998). S odium chann els as molecular

t a rget s for a nt iepilept ic drugs . Brai n Res. B rain Res. Rev. 26:16–28.

am an, I . M., Sprunger, L. K., Meisler, M. H., a nd B ean, B . P. (1997).

A lt ered s ubt hres hold s odium current s a nd dis rupt ed firing pa t -

t erns in P urkinje neurons of Scn8a m u t a n t m i ce . Neuron 19:881–891.

oja s , C. V. , Wa ng, J . Z . , Schw a t rz , L . S. , H offma n, E. P . , P ow ell ,

B. R., an d B rown, R. H., J r . (1991). A Met-to-Val mut at ion in t he

skeletal muscle Na ϩ alpha-subunit in hyperkalemic periodic pa-

ralysis. N a t u r e 354: 387–389.

uddle, F. H. , B a rt els , J . L . , B ent ley, K. L . , Ka ppen, C. , M urt ha ,

M. T., an d P end leton , J . W. (1994). Ev oluti on of H OX genes. A n n u .

Rev. Genet. 28: 423–442.

Rudolph, J . A., Spier, S. J . , By rns, G ., Rojas, C. V., B ernoco, D., a nd

Hoffman, E. P. (1992). Periodic paralysis in quarterhorses: A so-dium cha nnel mut a t ion dis s emina t ed by s elect ive breeding. N a t .

Genet. 2: 144–147.

Sa nga mes w a ra n, L . , Delga do, S. G. , Fis h, L . M . , Koch, B . D. , J a ke-man, L. B. , Stewart, G. R., Sze, P. , Hunter, J . C. , Eglen, R. M., andHerman, R. C. (1996). Structure and function of a novel voltage-gated, tetrodotoxin resistant sodium channel specific for sensoryneurons. J. B iol . Chem. 271: 5953–5956.

Sarao, R., Gupta, S. K., Auld, V. J . , and Dunn, R. J . (1991). Devel-opmenta lly regulated a lterna tive RNA splicing of ra t bra in sodiumchannel mRNAs. Nucleic Acids Res. 19: 5673–5679.

S a t o , C . , a n d M a t s u m ot o , G . (1 99 2). P r i m a r y s t r u ct u r e of s q u idsodium channel deduced from t he complementary DNA sequence.

Biochem. Biophys. Res. Commun. 186: 61–68.

Scha ller , K. L . , Krzemien, D. M . , M cKenna , N. M . , a nd Ca ldw ell ,J . H . (1992). Alternat ively spliced sodium channel tra nscripts inbra in a nd mus cle. J. N eur osci. 12: 1370–1381.

Smit h, R . D. , a nd Goldin, A . L . (1996). P hos phoryla t ion of bra insodium channels in the I-II linker modulates channel function in

Xenopus oocytes. J. N eur osci. 16: 1965–1974.

Smit h, M . R . , Smit h, R . D. , P lummer, N. W. , M eis ler , M . H. , a nd

Goldin, A. L. (1998). Functional analysis of the mouse Scn8a so-dium channel. J. Neurosci. 18: 6093–6102.

Souslova, V. A., Fox, M., Wood, J . N., an d Akopian, A. N. (1997).Cloning and characterization of a mouse sensory neuron tetrodo-t o x in -r e s is t a n t v ol t a g e -g a t e d s od i u m c h a n n e l g e n e Scn10a.

Genomics 41: 201–209.

Spa fford, J . D ., S pencer, A. N., a nd G allin, W. J . (1998). A puta tivevolt a ge-ga t ed s odium cha nnel a lpha s ubunit (P pSCN1) from t he

hydrozoa n jellyfi s h, Polyorchis penicil latus: St ruct ura l compa ri-s ons a nd evolut iona r y cons idera t ions . Bi ochem. Bi ophys. Res.

Commun. 244: 722–780.

S p r u n g e r , L . K . , E s ca y g , A. , Ta l l a k s e n -G r e e n e, S . , Al b i n , R . L . ,a n d M eis ler , M . H. (1998) G enera lized dys t onia a s s ocia t ed w it hm u t a t i o n o f t h e n e u r o n a l s o d i u m c h a n n e l Scn8a: R o l e o f t h e

modifi er locus Scnm1 on mouse chromosome 3. H um. M ol. G enet., in pres s .

Suzuki, N., a nd Ka no, M. (1977). D evelopment of action potential inla rva l mus cle fi bers in Dr osophila melanogaster. J. Cell . Physiol.

93: 383–388.

Ta t e, S. , B enn, S. , Hick. C. , Trezis e, D . , J ohn, V . , M a nion, R . J . ,Cos t iga n, M . , P lumpt on, C. , G rose, D. , G la dw ell , Z ., Kenda ll , G . ,Da le, K., B ountra , C. , an d Woolf, C. J . (1998). Two sodium chan-

nels contribute to the TTX-R sodium current in primary sensoryneurons. N at. N eur osci. 1: 653–655.

Ta ylor , C. P . , a nd Na ra s imha n, L . S. (1997). S odium cha nnels a nd

therapy of central nervous system diseases. Adv. Pharmacol. 39:47–98.

Th o m ps on , J . D . , H i g g i n s , D . G . , a n d G i b s o n , T. J . (1 99 4).

CL U STAL W: I mproving t he s ens it ivit y of progres s ive mult iples e q u en c e a l i g n m en t t h r o u gh s eq u e n ce w e i g h t in g , p os i t io n s-s p ec ifi c g a p p e n a l t i es a n d w e i g h t m a t r i x c h oi ce . N u cl ei c A ci d s

Res. 22: 4673–4680.

Toledo-Ara l, J . J ., Moss, B. L., H e, Z-J ., Koszowski, A. G ., Whisena nd,T., Levinson, S. R., Wolf, J . J ., Silos-Sa ntia go, I., Ha legoua, S., a ndMandel, G . (1997). Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neu-rons. Proc. N atl. A cad. Sci. U SA 94: 1527–1532.

Ts eng-Cra nk, J . , P olla ck, J . A. , Ha ya s hi, I . , a nd Ta nouye, M . A.(1991). Expression of ion channel genes in Drosophila. J. Neuro-

genet. 7: 229–239.

W a l l a c e , R . H . , W a n g , D . W . , S i n g h , R . , S c h e f f e r , I . E . , G e o r g e ,

A . L . , P hil l ips , H. A . , Sa a r , K. , R eis , A . , J ohns on, E. W. , Sut h-erla nd, G. R . , B erkovic, S. F. , a nd M ulley, J . C. (1998). Febriles eizures a nd genera lized epileps y a s s ocia t ed w it h a mut a t ion int h e N a ϩ-channel ␤1 s ubunit gene SCN1B . N at . G enet . 19: 366–370.

Wa n g , D . W ., Y a z a w a , K . , G e or g e, A . L . , J r . , a n d B e n n et t , P . B .

(1996a). Characterization of the human cardiac Na ϩ cha nnel mu-ta tions in the congenital Long QTsyndr ome. Proc. Natl. Acad. Sci.

U SA 93: 13200–13205.

Wang, Q., Shen, J ., Splawski, I., Atkinson, D., Li, Z., Robinson, J . L.,Moss, A. J . , Towbin, J . A., an d Keat ing, M. T. (1995). SC N5Amut a t ions a s s ocia t ed w it h a n inherit ed ca rdia c a rr hyt hmia , L ongQT syndrome. Cell 80: 805–811.

Wa ng, Q. , L i , Z ., Shen, J . , a nd Kea t ing, M . T. (1996b). Genomicorga niza t ion of t he huma n SCN5A gene encoding the cardiac so-dium channel. Genomics 34: 9–16.

Westenbroek, W. E., Merrick, D. K., and Catterall, W. A. (1989).

Differential subcellular localization of the R I a n d R II Na ϩ channelsubtypes in central neurons. Neuron 3: 695–704.

Wollner , D. A. , M es s ner, D. J . , a nd Ca t t era ll , W. A . (1987). ␤2

s u b un i t s of s od i u m ch a n n e l s f r o m v er t e br a t e b r a i n : S t u d i esw i t h s u b u n i t -s p ec ifi c a n t i b od i e s. J . B i o l . C h e m . 262: 14709–14715.

Ya ng, J . S. , B ennet t , P . B . , M a kit a , N. , George, A . L . , a nd B a rchi,R. L. (1993). E xpression of t he sodium channel ␤1 s ubunit in ra tskeletal muscle is selectively associated with the tetrodotoxin ␣

subunit isoform. Neuron 11: 915–922.

1999 evolution and diversity of mammalian sodium channel genes.pdf

Documents

neuronalcircuitrymechanismsregulatingadult mammalian

mammalian circulatory system

the mammalian eye

plant and mammalian tissue culture transfection and...

mammalian digestive tracts

35 mammalian heart

mammalian glycosidases

mammalian 2

mammalian eye

mammalian nutrition ocr mammalian physiology and behaviour...

mapping of voltage sensor positions in resting and ... ·...

s1172, sodium bisulfite, granular, fcc - spectrum...

mammalian reproduction

mammalian classification

mammalian senses

mammalian toxicology - university of...

mammalian anatomy

pa mammalian predators

mammalian characteristics:

mammalian neuroanatomy