Download An isoform of the cGMP-gated retinal photoreceptor channel gene

Biochemical SocietyTransactions ( 1 993) 21
An isoform of the cGMP-gated retinal
photoreceptor channel gene expressed in the
sinoatrial node (pacemaker) region of rabbit
heart
SUKHINDER P. HUNDAL', DARIO DIFRANCESm,
MATTE0 MANGOW. WILLIAM J. BRAMMARl
and EDWARD C. CONLEY'
llCllJoint Laboratory, Department of Biochemistry, Universityof
Leicester, Leicester, L E I 7RH and2Dipartimentodi Fisiologia.
University of Milan, Italy.
Spontaneous electrical activity of the mammalian heart
arises from specialized pacemaker cells of the sino-atrial
(SA) node. These cells have previously been shown to
exhibit four voltage-sensitive currents (ICaT. ICaL. IK, If)
in addition to a 'background current (IbNa), a K+ current
activated by acetylcholine (IKACh). and the electrogenic
currents of the Na+, K+ ATPase and the Na+-Ca2+
antiporter [l]. Of these components, it has been suggested
that the inward, hyperpolarization-activated, non-specific
cationic conductance (described by if) may contribute to
development and modulation of a slow 'pacemaker'
depolarization during the diastolic phase. Adrenaline shifts
the voltage-activation curve of if in a positive direction; this
action is mediated by CAMP, which directly activates if by a
mechanism independent of phosphorylation [2]. We
therefore became specifically interested in the expression of
cyclic-nucleotide-gated ion channel genes within the cardiac
SA node.
A total of thirty-eight individual rabbit sinoatrial node
preparations were collected by a micro-dissection procedure
followed by rapid freezing in liquid nitrogen. Particular
care was taken to avoid atrial tissue and contamination by
RNAses. Typically, the sinus node preparations yielded -5
pg polyA(+) mRNA / g tissue, and 5pg was sufficient to
generate a directional representative cDNA library in a
hZAP-XR cloning vector (Stratagene Inc.). We designed a
set of non-degenerate PCR primers based on conserved
sequences between the published bovine rod cGMP-gated
channel [3] and rat olfactory CAMP-gated channel [41. The
riboprobe used for screening encompassed sequences
encoding the functional regions for the ion-selective pore
and the c clic nucleotide binding domains. Approximately
2 x 10 clones were screened under low stringency
conditions by conventional methods.
d -
From the limited number of hybridizing clones sequenced
thus far (six), the majority do not show significant
homology to any previously-reported sequences in the
current release of the Genbank sequence database.
However, one clone showed striking homology to the
protein coding and 3' untranslated regions of the cGMPgated non-specific cationic channel expressed in the plasma
membranes of vertebrate rod photoreceptors. Alignment of
the existing sequence with the human and mouse cGMPgated channel isoforms (Figure 1) indicates that a rabbit
isoform of the photoreceptor channel is expressed in the
highly-localized region of heart used for construction of the
original cDNA library. This finding is intriguing for a
1 19s
number of reasons - First, to our knowledge, there have
been no previous reports of a role for such a channel in the
SA node region or indeed any cardiac subregion.
Possession of cyclic nucleotide binding sites which directly
activate the channel may however provide a mechanism for
fast modulation by neurotransmitters in the absence of
phosphorylation (by analogy to that shown for the if
channel, see above). Secondly, the cyclic nucleotide
sensitivity of such channels probably necessitates the
coupling of appropriate receptors/second messenger systems
to gate the channel in the heart; these are unlikely to be
related to those types co-expressed in the photoreceptor.
Thirdly, the gene sequence for the human cGMP-gated
channel has been determined [5] and contains many potential
splice sites. The presence of such sequences raises the
possibility of tissue-specific splice variants although no none
have been observed to date. We are presently determining
the entire sequence of the rabbit SA node isoform and
progressing to functional expression studies in mammalian
cells to determine if any structural or functional differences
exist between the rabbit SA node isoform and other related
sequences.
[ l ] Brown, A.M. (1990) Am. J . Physiol259, H1621-H1628.
[2] DiFrancesco, D. and Tortora, P. (1991) Nature (London) 351,
145-147.
[3] Kaupp, U.B., Niidome, T., Tanabe, T., Terada. S., Bonigk,
W., Stiihmer., W., Cook, N., Kangawa, K., Matsuo, H., Hirose,
T. and Numa. S. (1989) Nature (London) 342,762-766.
[4] Dhallan, R.S., Yau,K., Schrader, K. and Reed, R. (1990)
Nature (London) 347, 184-187.
[5] Dhallan, R.S., Macke. J.P.,Eddy, R.L., Shows, T.B., Reed,
R.R., Yau, K-W. and Nathans, J. J. Neuroscience 12, 32483256.
Human
L y s Asp L y s G l u L y s L y s L y s Lyo Glu G l u L y s Ser L y s Asp L y s (147)
I
Rabbit AAA GAG AAA GAG AAG AAA AAG AAA GAG GAG ACA AAG AAA GAA AAG
Rabbit L y s G l u L y s G l u L y s L y s L y s L y s G l u G l u Thr L y s L y s G l u L y s
I
Mouse
L y s G l u L y s G l u L y s L y s L y s L y s G l u G l u L y s T h r L y s G l u Lya (139)
Human
L y s G l u His His L y s L y s G l u V a l V a l V a l Ile Asp P r o Ser G l y (162)
I
Rabbit AAA GAR GAG GAG I\py; AAA GAA GTC ATG ATT ATT GAT CCC TCA GGA
Rabbit L y s G l u G l u G l u L y s L y s G l u V a l M e t Ile Ile Asp P r o Ser G l y
I
Mouse
L y s G l u G l u G l u L y s L y s G l u V a l V a l V a l Ile Asp P r o Ser G l y ( 1 5 4 )
Human Asn T h r T y r T y r Asn T r p Leu P h e C y s Ile T h r Leu P r o V a l Met (177)
I
Rabbit AAC ACA TAT TAC A&C TGG CTG TTT TGT ATC RCT TTA CCT GTG AT2
Rabbit Asn T h r T y r T y r Asn T r p Leu Phe C y s Ile Thr Leu P r o V a l Met
I
Mouse Asn T h r T y r T y r Asn T r p Leu P h e C y s Ile T h r Leu P r o V a l M e t (169)
Human
T y r Asn T r p T h r Met V a l Ile A l a Arg A l a C y s P h e Asp G l u Leu (192)
I
Rabbit TAC AAC TGG ACT ATG ATT ATT GCA M A GCC TGT TTT GAT GAR CTT
Rabbit T y r Asn T r p T h r Met Ile Ile A l a Arg Ala Cys Phe Asp G l u Leu
I
Mouse
T y r Asn T r p T h r Met Ile Ile A l a Arg A l a C y s Phe Asp G l u Leu (184)
Human
G l n Ser Asp T y r Leu G l u T y r T r p Leu Ile Leu Asp T y r V a l Ser (207)
I
Rabbit CAR TCT GAT TAC CTA GAA TAT TGG CTC ATT TGT GAC TAC TTG TCA
Rabbit G l n Ser Asp T y r Leu G l u T y r T r p Leu Ile Cys Asp T y r Leu Ser
I
Mouse
G l n Ser Asp T y r Leu G l u T y r T r p Leu Ile P h e Asp T y r V a l Ser (199)
Human
Asp Ile V a l T y r Leu Ile Asp Met P h e V a l Arg Thr Arq Thr G l y (222)
I
Rabbit GAT ATA GTC TAT CTT GTT GAC ATG TTT GTA CWL ACA CGG ACA GGT
Rabbit A s p Ile V a l T y r Leu V a l Asp t4t P h e V a l Arg T h r Arg T h r G l y
I
Mouse
Asn V a l V a l T y r Leu Ala Asp M e t P h e V a l Arg Thr Arg Thr G l y (214)
Figure 1: Comparison ofpartial nucleotide sequence and predicted
amino acid sequences of the human and mouse cCMP-gated
photoreceptor channels with the rabbit isoform expressed in SA
nodal region. Amino acid residue numbers are indicated in brackets.