4.3. PLAN DE MANEJO AMBIENTAL
4.3.2. Programa de medidas preventivas y/o correctivas
A tenth m em ber of the voltage-dependent calcium channel a j subunit gene fam ily, a n , was identified by database searching using a m and a m cD N A s against the hum an genom ic sequence database. Preliminary analysis shows the novel gene to have high predicted am ino acid sequence homology to both a m and a m subunits suggesting it to be a m em ber of the T-type class (Perez-Reyes et al., 1998a).
T a b l e 1.6.2 P r o p e r t i e s , p r e d o m in a n t s i t e o f e x p r e s s io n a n d h u m a n m ap p o s i t i o n s o f t h e VOLTAGE-DEPENDENT CALCIUM CHANNEL a , PORE FORMING SUBUNITS.
Subunit Locus Type Predominant siteof expression Human chromosomal
localisation
«IS C A C N A J S H V A L -type Skeletal M u scle Iq 3 1 -q 3 2
«1A C A C N A I A H V A P/Q -type Brain 1 9 p l3 .3
O^IB C A C N A I B H V A N -type Brain 9 q 34
o^ic C A C N A I C H V A L -type
H eart/Lung/
Sm ooth M u scle 1 2 p te r -p l3 .2
CtlD C A C N A I D H V A L -type Brain/Pancreas 3 p l4 .3
<^1E C A C N A I E H V A R -type Brain/Heart Iq 25-q 31
ttiF C A C N A I F H V A L -type R etina X p 11.23
O^IG C A C N A I C L V A T -type Brain 17q22
O^IH C A C N A I H L V A T -type Heart 1 6 p l3 .3
a n C A C N A l l L V A T-type Brain 2 2 q l2 .3 - 1 3 .2
1 .6 .4 Th e aiÔ s u b u n it
The a z a n d Ô subunits are encoded by a single gene producing a 1091 am ino acid peptide w hich was cloned in humans using homologous cD N As previously isolated in the rabbit (W illiam s et at., 1992b). Once synthesised the precursor undergoes proteolytic cleavage near the carboxyl terminus to produce separate az and 6 subunits
that becom e covalently linked via disulphide bonds to form the m ature « 2 6 com plex.
Secondary structure analysis of the a ib subunit predicted three transm em brane dom ains (W illiam s et a l , 1992b). Antibody staining later show ed that only one of these is actually used to anchor the bound extracellular % peptide to the lipid m em brane (reviewed in Perez-Reyes and Schneider, 1995). FISH analysis of OC2 8 assigned the hum an locus, CACNA2, to chromosom e 7 q l 1.23-q21.1 (lies et a l ,
1994).
The a2 0 com plex has been shown to be expressed in a variety o f tissue such as
skeletal m uscle, heart, aorta, lung and brain and to be a com ponent of the N -type brain and L-type skeletal and cardiac channels. The human a j has also been shown to be alternatively spliced for three exons producing up to five alternative tissue specific transcripts (Table 1.6.3) (W illiams et a l, 1992b); and (review ed in Perez-R eyes and Schneider, 1995). Although the physiological role is not fully understood, expression studies using different a i subunits with various splice form s o f the 0126 subunit have
suggested that it may be involved in controlling the spatial arrangem ent and stability of calcium channels in the cell membrane. The major role o f the 0628 subunit is
thought, how ever, to be in altering the kinetics of voltage dependence o f activation and inactivation (reviewed in Perez-Reyes and Schneider, 1995).
1 .6 .5 Th e Y SUBUNIT
Like m any of the voltage-dependent calcium channel subunits, the yi peptide and cD N A sequence was originally characterised in the rabbit (Bosse et a l , 1990), and only later in human. The 25kD subunit has four putative m em brane spanning dom ains (review ed in Perez-Reyes and Schneider, 1995) with both intracellular am ino and carboxyl term inals.
G enom ic structure analysis of the C AC N G l gene showed that the 1.3kb cD N A is coded for by four exons spread over a genomic distance o f 12.5kb on chrom osom e 17q22-24 (Powers et a l , 1993). The resulting 222 amino acid yi peptide is alm ost exclusively expressed in skeletal muscle (Bosse et a i, 1990). Expression studies show the Yi to have no direct effect on the cardiac a jc pore form ing subunit. H ow ever, in the presence o f the p subunit, yi increases peak currents and the rate o f channel activation and inactivation (W ei et a l , 1991).
Sequence analysis of the second member of the y subunit fam ily, C A C N G l, predicts an open reading fram e coding for a 36kD peptide, 100 am ino aeids longer than
C A C N G l with only 25% identity and 38% similarity over 200 am ino acids (Letts et al., 1998). H ydrophobieity plots however, predict the two peptides to exhibit m ueh higher degrees of structural homology suggesting that even though the peptide sequence is substantially divergent, its functional role is eonserved. C o-expression studies o f y% with ajA, Pib and ŒzÔ showed it to be able to shift the voltage dependence o f aetivation, like the skeletal y, subunit, significantly reducing the channel availability at normal resting potential (Letts et al., 1998).
U nlike skeletal y%, the y% subunit is only expressed throughout the brain w ith highest levels being detected in cerebellum, olfactory bulb, thalamus and hippocam pus (Letts
et al., 1998).
The results of electrophysiologieal studies on both yi and y% suggest that the m ain role of these subunits is in stabilising ehannel inactivation resulting in a net reduction in channel availability at resting potential (Wei et al., 1991; Letts et al., 1998).
Ta b l e 1.6.3 Hu m a n ex p r e ssio n pa tte rn o f v o l t a g e-d e p e n d e n t c a l c iu m c h a n n e l a u x il ia r y
SUBUNIT ISOFORMS AND SPLICE VARIANTS.
Subunit Isoforms Predominant site of expression Reference
a i ô Ct2a Skeletal M u scle (W illia m s et al., 19 9 2 b )
0C2b Aorta, Brain (W illia m s e t al., 1992b )
C^2c) CC2d, CC2e Aorta (B rust e t a l , 1993)
Yi - Skeletal M u scle (P ow ers et al., 1 992b )
Ï2 - Brain (L etts et al., 1 9 9 8 )
P. Pia Skeletal M u scle and Heart (P ow ers e t a l . , 1992b )
Plb Brain and Heart (P ow ers et al., 1992b )
P ic Brain and Heart (P ow ers e t a l . , 1992b )
P2 P2a Brain, Heart, Lung (P erez R e y e s et a i , 1 9 9 2 )
P2b Brain, Heart, Lung (P erez R e y e s et a i , 1 9 9 2 )
P2c Brain (P erez R e y e s et al., 1 9 9 2 )
P3 - Brain, Ovary, C olon (C o llin et al., 1 9 9 4 )
1 .6 .6 Th e (3 s u b u n i t, g e n e s a n d s p l ic ev a r ia n t s
V arious form s of the human voltage-dependent calcium channel P subunit are currently know n to be encoded by four highly hom ologous genes C A C N B l, CACNB2, CACNB3 and CACNB4 (Ruth et al., 1989; Rosenfeld et al., 1993; Gregg et al., 1993b; Collin et al., 1994; Taviaux et al., 1997; Escayg et a l , 1998).
The prim ary sequence and predicted secondary structure o f the four fam ily m em bers appears to be highly conserved containing four to six a-h elices (Ruth et al., 1989). H ydrophobieity plots reveal regions of the peptide to be highly charged indicating that although previously shown to be localised on the surface (Chien et al., 1995), the subunit is probably not im bedded in the lipid m em brane (review ed in Perez-R eyes and Schneider, 1995). Tw o p subunit family members, pi and P2 have been shown to
exhibit splice variation (Perez Reyes et al., 1992; W illiam s et al., 1992b). E xpression studies show that each subunit and splice variant are localised to specific tissue types (Table 1.6.3). One of the subunits, pi, has been shown to be post-translationally m odified by PKA phosphorylation (Ruth et al., 1989), and one, P%, contains a consensus palm itoylation site (Collin et al., 1994).
The function of the p subunit is yet to be fully understood. Co-expression of individual subunits with a i peptides in Xenopus oocytes has shown them capable of dram atically altering channel biophysical characteristics, typically by increasing peak currents and accelerating activation and inactivation kinetics. A detected increase in num ber o f available channels at resting potential in the presence of the P subunit suggests a role in the m em brane localisation of the (X| pore (review ed in Perez-Reyes and Schneider, 1995) (See also section 1.6.7.II.ii).