Distribution of Voltage Gated K“ Channel B
Subunits in the Optic Lobe of Adult, Hatchling,
and Embryonic Squid, Loligo opalescens
Tammy Wang
Hopkins Marine Station
Stanford University
Spring 1997
Professor William F. Gilly
ABSTRACT
Kyß subunits associate with a subunits and cause changes in both properties
(Kyß1) and expression level (Kyß2) of Ky potassium channels. Kyß1 has been noted to
Increase channel inactivation rates when co-expressed with Kva. Increases in
glycosylation efficiency and surface protein expression of a subunits have been
observed in the presence of Kyß2. A partial KVB CDNA clone (Sgß) was isolated from a
squid stellate ganglion CDNA library and tentatively identified as a 82-type. In situ
hybridization was used to examine Sgß mRNA distribution in adult, hatchling, and
embryonic squid. In optic lobes, RNA probe specific for Sgß preferentially labeled
cellular layers containing somata of secondary and tertiary visual neurons, bipolar
cells, and large amacrine cells. Based on grain density observations, Sgß was present
in nearly all medullar cells of post-embryonic squid. In stage 29+ embryos, Sgß grain
density was noticeably lower than in adults and hatchlings. Kyß subunits may be
expressed in different cells at various levels during development, adding another level
of regulation and possibly playing an important role in increasing Ky channel diversity.
INTRODUCTION
Voltage gated potassium channels are a diverse group of membrane spanning
proteins with functions ranging from regulation of action potentials to cardiac pace¬
making (Connor and Stevens, 1971; Byrne, 1980; Rogawaski, 1985; Hille, 1991) to
hormone secretion (Dubois and Rouzaire-Dubois, 1993). Such diversity of function is a
result of structural flexibility and variety. Channels are composed of associated a and
B-subunits. Ky channels are tetrameric proteins, consisting of 4 a-subunits which must
be from the same subfamily (Covarrubias et al. 1991; Li et al. 1992; Sheng et al. 1993:
Wang et al. 1993; Deal et al. 1994). Some Ky families also associate with cytoplasmic
B-subunits thus increasing structural diversity (Pongs, 1995).
In the past, most research on Ky channels has focused on the a-subunit.
However, examination of the a-subunit alone may present an incomplete picture of true
channel function since both a and B-subunits are present in vivo. Kyßs are divided into
two classes by their effects on channel function when co-expressed with a-subunits.
Kyßs are cytoplasmic proteins which have been found to interact specifically with the
NAB domain of KV1 a-subunits, a region which directs the different a-subunits to form
heterotetramers (Yu et al. 1996). The interaction of a and ß in the Kvl family does not
seem to require the N-terminus of the B-subunit (Nakahira et al. 1996).
Although the in vivo function of B-subunits alone is unknown (Yu et al. 1996), the
B-family has been found to have significant effects when co-expressed with a¬
subunits. Five B-subunit CDNAs, arising from three related genes, have been isolated
from mammalian heart and brain (Rettig et al. 1994, Majumder et al. 1995; Morales et
al. 1995, England et al. 1995a; McCormack et al. 1995; Heinemann et al. 1995:
England et al. 1995b). Kyß1 and Kyß3 are known to increase inactivation rates of Kyl
channels by using a globular domain at the amino terminus similar to the inactivation
"ball" found in certain a-subunits (Hoshi et al. 1990; Rettig et al. 1994). Recent
experiments with ß3 suggest that B-subunits may act selectively, only associating with
some a subunits (Majumder et al. 1995; Morales et al. 1995). Kyß2 subunits, the most
abundant of the B-subunit isoforms in the mammalian brain (Rhodes et al. 1995:
Rhodes et al. 1996), appear to lack the amino terminus which functions as the
mactivation "ball" of B1 and ß3. Instead, 82, which shares -90% sequence homology
with B1 and 83 (Rettig et al. 1994), has been found to play a crucial role during K
channel biosynthesis by promoting cotranslational N-linked glycosylation of the initial
KV1.2 polypeptide, increasing stability of a-B complexes, and augmenting efficiency of
a-subunit expression (Shi et al. 1996). This effect is, in part, believed to involve 82
assisting in the proper folding of a-subunit polypeptides mediated through the
cotranslational interaction of ß2 with the a-subunit N-terminal cytoplasmic domain.
A partial B CDNA clone (Saß) has been isolated from a squid stellate ganglion
CDNA library and includes 3' coding and untranslated regions. In this study, we used in
situ hybridization techniques to search for Sgß mRNA in the optic lobe of adult.
hatchling, and embryonic squid. The optic lobe, a football-shaped structure adjacent to
each eye, is the primary region processing visual information from the eye. Divided
into three main regions, the two optic lobes are located on either side of the brain.
Secondary and tertiary visual cells are present in the optic lobe, along with numerous
other types of interneurons.
Although the partial Sgß CDNA clone is missing the 5' end required for
unambiguous class identification, alignment with known ß sequences has led us to
believe that Saß may be a member of the B2 family. Using in situ hybridization, we
were able to locate Saß mRNA in various layers of the adult squid optic lobe. We also
searched for ß in the different stages of development to obtain a qualitative idea of
distribution patterns.
MATERIALS AND METHODS
Tissue preparation
Live adult squid (Loligo opalescens) were collected in Monterey Bay, California.
and were held in tanks with circulating sea water at ambient temperatures (13 - 15 °c).
Embryos were from egg capsules laid by the captured squid. Egg capsules, once
collected, were kept until hatching in 20 gallon circular tanks with constant flow-through
seawater at ambient temperatures. Hatchlings (38 days old) used in this experiment
were kept in flow-through tanks and raised on a mixed diet (fed both Artemia and
copepods)
Tissues used for these experiments were dissected and fixed for approximately
one hour in an ice-cold 4% paraformaldehyde solution in PBS (130 mM Nacl, 7 mM
NazHPOz, 3 mM NaHzPO4, pH 7.0) directly after decapitation. Only the heads of the
embryos were used. Hatchlings had their mantles cut open to facilitate perfusion of
Polyfreeze. Tissue was then washed for 3 x 5 minutes in PBS before being
cryoprotected 2 x 40 minutes in PBS with 20% sucrose. All procedures were done on
ice (4 °C). Tissues were blotted dry and coated with Polyfreeze (Polyscience) before
being embedded in Polyfreeze in gelatin capsules over dry ice. 8 - 12 micron sections
were cut in a cryostat and dried onto silanated slides. Slides were then washed in PBS
followed by graded dehydration through ethanol (50%, 70%, 95%, 100%). One slide
per set was stained with multiple stain (toluidine blue and basic fuchsin) and air dried
for 2 hours before mounting with Permount and xylene. After drying in the hood, slides
were either used for in situ hybridization or stored with dessicant at -70 °c.
Probe design and construction
Antisense probe (- 200 bp) was synthesized from a partial beta CDNA clone in
Bluescript KS+ phagemid (pBeta 3'-)after being linearized with Xmal. (Figure 1)
Transcription with T7 RNA polymerase was done using a Riboprobe kit (Promega) in a
5 ml reaction volume with 2S labeled UTP (20 uCilml). Sense probe (- 180 bp) was
transcribed from the same plasmid with T3 RNA polymerase after linearizing with BstxI.
Labeled probes were purified in Nu-Clean spin columns (IBI) and used in hybridization
buffer (62.5% deionized formamide, 12.5% dextran sulfate, 375 mM Nacl, 1.25X
Denhardts, 12.5 mM Tris pH 8.0, 1.25 mM EDTA) at a final concentration of about 5 x
106 cpm/ml.
Hybridization procedure:
The hybridization procedure used was described by Liu and Gilly (1995) and
adapted from Simmons et al (1989). Slides were incubated in proteinase K, rinsed in
triethanolamine, soaked in acetic anhydride, and washed in 2X SSC. Before probe
application, mounted tissue was dehydrated in ethanol, allowed to dry, and vacuum
desiccated for 2 hours to prevent tissue loss during hybridization. Probe was heated to
65 °C for 10 minutes. Approximately 300 - 400 ul of heated probe was then applied to
slides. A slightly-oversized piece of parafilm was placed on the slide, being careful to
avoid trapping bubbles. Slides were stored overnight in small, loosely-covered plastic
containers which were subsequently placed in a larger tightly sealed humid container at
60 °C. Parafilm coverslips were soaked off and slides washed in 4X SSC (0.15 M.
Nacl, 0.015 M sodium citrate). This was followed by treatment with RNase A and
washed in sequentially diluted solutions of SSC. 1 mM DTT was included in all SSc
washes. Slides were dehydrated in ethanol (50% and 70% EtOH made with SSC and
DII) and dried in a hood. Slides were coated with NTB-2 emulsion (Kodak) and stored
in light-tight containers for 7 - 10 days at 4 °C before development (4 min in Kodak D¬
19). After fixation (Kodak Rapid Fixer), slides were washed for -1 hour in running tap
water. They were then washed in acetate buffer (0.54% sodium acetate, 0.96% acetic
acid) and stained in 0.5% cresyl violet in acetate buffer for 4 minutes before being
processed through 50%, 70%, 95%, 100% EtOH and xylene. Finally, slides were
mounted with Permount and xylene and dried in the hood.
Quantification of signal density
Black and white video images were taken of slides. An imaging program (NIH
lmage 1.60/ppc) was used to capture frames from Hi-8 video images. Number of grains
was counted in randomly selected areas. The number of grains per unit area was
calculated by the imaging program. P-values less than 0.01 from t-tests comparing
signal densities were considered significant.
RESULTS
Optic Lobe Structure in Adult Squid
General histology corresponds to that reported by Young (1974) (Appendix 1).
The outer granule layer (arrowhead; Figure 2) is about 70 microns thick and contains
several different types of cells. Secondary visual cells and large amacrine cells are
concentrated in the outer half of the outer granule layer and small amacrine cells are
localized to the inner half. The two granule layers are separated by the plexiform zone
(asterisk; Figure 2) which contains mostly axon bundles, dendrites, and glia. The inner
granule layer has small amacrine cells in the outer half and bipolar cells in the inner
region. The majority of the optic lobe consists of the medulla which contains an
interwoven network of cells, including tertiary visual cells, separated by areas of
neuropil.
In situ hybridization in adult optic lobe
In situ hybridization of sections with Saß antisense probe showed high leyels of
signal density in the peripheral region of the outer granule layer (Figure 3a). Labeling
with the sense probe did not show labeling above background levels (Figure 3b).
Hybridization of sections with Sgß antisense probe showed various signal
densities in different cell layers within the same tissue sample. Labeling in the
peripheral outer granule layer where secondary visual cells and large amacrine cells
predominate was significantly greater than in the interior of the outer granule layer.
containing mostly small amacrine cells (Figure 4a). Labeling in the inner part of the
Inner granule layer, with bipolar cells, was significantly greater than in the outer region
of the inner granule layer, where small amacrine cells are located (Figure 4b). Signal
densities in cells of the medulla were greater than those found in the surrounding
neuropil. (Figure 4c)
The histogram in Figure 5 describes the quantification of signal densities in each
cell layer labeled with antisense probe compared to that labeled with sense probe.
Grain counts in layers containing secondary and tertiary visual cells and bipolar cells
were significantly greater than those seen in small amacrine cell layers. Also, grain
counts were significantly greater in cellular regions of the medulla compared to neuropil
regions, indicating lack of Saß mRNA in axons, dendrites, and glia.
Differential expression of B during development
Cross-sections were taken through the horizontal plane of a 38 day old
hatchling. Basic structure of the optic lobe was similar to that of the adult. However.
the plexiform zone (asterisk; Figure 6) was much smaller and neuropil did not constitute
as much of the medulla in comparison to the adult. Rather, cell clumps filled most of
the medulla. Amacrine cell layers were proportionately smaller, taking up less than a
quarter of each of the granule layers.
Late (294) stage embryo (Segawa et al. 1988) heads were also sectioned.
Again, basic histology followed adult optic lobe. However, the plexiform zone was even
smaller and in some areas, the two granule layers did not appear to be separated
(Figure 7). Almost no neuropil was present and we were unable to differentiate
between the different cells of the granule layers.
Labeling in the peripheral outer granule layer seems to increase with
developmental stages (Figure 8). However, it was not possible to compare signals
quantitatively between developmental stages because they were carried out as
separate experiments.
DISCUSSION
We constructed our RNA probe from a partial sequence obtained from the CDNA
library of the stellate ganglion. Although an RNase protection assay showed that Saß
was expressed in the stellate ganglion, we were unable to locate significant amounts of
Saß in this tissue by in situ hybridization. Sgß may be present in low abundance in the
stellate ganglion.
Because we lacked the N-terminus which is the primary determinant of the type
of ß subunit, we compared our sequence with those in the literature in an attempt to
classity Saß. Aside from the N-terminal, differences between rat Kyß1 and rat Kyß2 are
primarily scattered point differences. (Figure 9) However, many of these point
differences are conserved when comparing rat and bovine 82. Our partial clone
matched the rat and bovine ß2 in 14 sites where rat and bovine 82 were identical but
different from rat ß1. The opposite case, where our Sgß matched rat 81 where rat and
bovine B2 were the same, only occurred at 5 sites. Thus, it appears to be likely that
Saß belongs to the 82 family.
Our in situ hybridization results seem to indicate that in adult squid optic lobe.
different amounts of Saß mRNA are found in different cells. Sgß mRNA appears to be
found in larger amounts in the regions where secondary visual cells and bipolar cells
are located. In contrast, low levels of Sgß mRNA are present in the regions
surrounding the plexiform zone which contains mostly small amacrine cells, High
signal density was found in some of the cells in the medulla, these possibly being the
tertiary visual cells. Saß mRNA did not seem to be present in the neuropil and
plexiform layers which lack cell bodies
The neurons in the optic lobe process visual information from the eye. The
primary visual cells in the eye synapse onto the secondary visual cells in the optic lobe
where high density signals were seen. Bipolar cells act as interneurons which transmit
information from the secondary visual cells to the tertiary visual cells, the primary
output neurons from the optic lobe to the brain. In each of these cell types a high
density of labeling was found, relative to the control sections, neuropil, and plexiform
zones.
Based upon our results, Saß appears to play an important and specific function
in squid optic lobe cells. Although we cannot determine the role that Sgß plays until we
obtain the full sequence and co-express it with a subunits, the presence of B subunits
indicated the potential for regulation of potassium channels in certain optic lobe cells.
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FIGURE LEGENDS
Figure 1 pBeta3'¬
Plasmid used in probe preparation. To obtain antisense mRNA, primarily in the
3 Untranslated region (UTR), plasmid was cut with Xmal and transcribed with T7 RNA
polymerase. For sense mRNA, used in controls, plasmid was cut with BstXl and
transcribed with T3.
Figure 2 Adult optic lobe
Section was stained with cresyl violet which only stains cell bodies. Scale bal
equals 150 microns. Outer granule layer (arrowhead) is separated from the inner
granule layer by the unstained plexiform zone (asterisk) which lacks cell bodies and
contains only axons and dendrites. Medulla is the region containing the islands of cells
separated by neuropil.
Figure 3 Outer granule layers labeled with both antisense (a) and sense (b) mRNA
probes
Scale bars equal 50 microns. Plexiform zone is labeled with a P, and outer
granule layer periphery is labeled with an O. Signal density is much higher in tissue
labeled with antisense mRNA probe than tissue labeled with sense probe.
Figure 4 In situ hybridization of adult optic lobe
Scale bar equals 30 microns.
(a) Outer granule layer of adult
Peripheral region is at top of picture. Higher concentrations of signals are
present at the periphery, where secondary visual cells are located, compared to the
Inner region where small amacrine cells are present.
(b) Inner granule layer of adult
Unstained portion at top is plexiform region. Periphery of this layer of cells
contains small amacrine cells and signal density is low. High grain density is present in
the lower portion of the layer where bipolar cells are located.
(c) Adult Medulla
Regions surrounding the islands of cells are the neuropil, containing axons.
dendrites, and glia. Higher levels of mRNA appear to be present in cell bodies than the
neuropil.
Figure 5 Histogram showing quantification of signal densities in adult granule
layers and medulla
Saß signal density was found to vary in different adult squid optic lobe cell
layers. Sense levels were consistently low in all cell layers. Density levels were low in
layers containing small amacrine cells (inner outer granule layer and outer inner
granule layer), but high in layers with visual and bipolar cells. (Antisense: Outer outer
granule layer: n = 8; mean + of grains/um“ = 0.0388; s.d. = 0.0154. Inner outer granule
layer: n = 8; mean F of grains/um“ = 0.0127; s.d. = 0.00243. Outer inner granule layer
n = 6; mean + of grains/um“ = 0.00813; s.d. = 0.00332. Inner inner granule layer: n=
6; mean + of grains/um“ = 0.0182; s.d. = 0.00377. Medulla: n = 11; mean tt of
grainslum“ = 0.0284; s.d. = 0.00562. Neuropil: n = 11; mean 4 of grains/um? -
0.00845; s.d. = 0.00218. Sense: Outer outer granule layer: n = 4; mean tt of
grains/um“ = 0.00975; s.d. = 0.00437. Inner outer granule layer: n = 4; mean 4t of
grainslum“ = 0.00674; s.d. = 0.00198. Outer inner granule layer: n = 4; mean tt of
grainslum“ = 0.00714; s.d. = 0.00230. Inner inner granule layer: n = 4; mean 4t of
grainslum“ = 0.00871; s.d. = 0.00197. Medulla: n = 4; mean + of grains/um? = 0.0114;
s.d. = 0.00121. Neuropil: n = 4; mean 4 of grains/um? - 0.00882; s.d. - 0,00277.)
Figure 6 Hatchling granule layers
Plexiform zone is labeled with an asterisk and medulla is labeled with an arrow.
Scale bar equals 30 microns. Section was stained with cresyl violet and labeled with
antisense Saß RNA probe. Inner and outer layers are separated by the plexiform zone
Figure 7 Stage 29+ embryo granule layers
Scale bar equals 30 microns. Outer granule layer is surrounded by a layer of
tissue around the optic lobe and is labeled with an asterisk. Labeling appears to be
localized to the peripheral outer granule layer. Inner granule layer (arrow) is beneath
the plexiform zone.
Figure 8 Comparison of (a) adult, (b) hatchling, (c) embryo outer granule layers
Scale bars equal 30 microns. Region just outside the outer granule layer is
labeled with an O, and P denotes the plexiform zone. Labeling density appears to
Increase in the periphery of the outer granule layer through development.
Figure 9 Beta Sequence
Alignment of Saß with rat ß1 and 82 and bovine B2 provide evidence that Saß
belong to the B2 family. Dashes represent the same residue as in the first line (rat
KVB1). Residues are boxed where Saß is the same as rat and bovine 82 but different
from rat B1. The underlined region was the 5' end of our probe which continued into the
untranslated region.
Appendix 1 Young's schematic
This schematic represents the optic lobe of an adult squid. The top most layer is
the outer granule layer, containing large and small amacrine cells, and secondary
visual cells. This is separated from the inner granule layer by the plexiform zone which
consists mostly of axons and dendrites. Bipolar cells and small amacrine cells are
found in the inner granule layer. The majority of the optic lobe consists of the medulla
which contains tertiary visual cells and other cell bodies.
Figure 1
T3A
&am
—
Xmal
BstXI

180 bp
- 200 bp
pBeta3'-
Bluescript KS+
3'UTR
Beta coding region
2T7
Figure 2


Figure 5
Beta Signal Density Varies in Different
Adult Squid Optic Lobe Cell Layers
0.04
- 0.03
00
0 +

OOGL
IOGI
OIGL
iIGL Med. Cells Neuropil
Cell laye
OOGL = outer outer granule layer
IÖGL = inner outer granule layer
OIGL = outer inner granule layer
IIGL = inner inner granule layer
Med. Cells = cells in the medulla
HAntisense
Sense
Figure 6
Figure 7

Figure 9
rat Kv BI AMYWGTSRWSAMEIMEAYSVAROF
rat Kv 32 -
— — — —
- - S.
— — — —
bov Kv ß2-------- - - S- - - - - - - - - - - - -
sq Kv ß2 CXG - - - - - - PR - - - - CCA - - - - -
rat Kv Bl NMIPPVCEQAEYHLFOREKVEVOL
rat Kv 82 ------
--  - -- ----
bov Kv 82 - |  | — - -  | - - - --- |  | - - - - - --
s Kv 2 - - - -  --- -
NMH -D---LHM
rat Kv B1 PELYHKIGVGAMTWSPLACGIISG
rat Kv ß2 - - - F - - - - - - - - - - - = = = = = = = V -
bov Kv ß2 - - - F - - - - - - - = = = = = = — — — = = V -
sq Kv B2 9D I - 9 - - - - - T T - - - - - S - - LT -
rat Kv BI KYGNGVPESSRASLKCYQWLKERI
rat Kv 82 - -Ds- I-P------G-
D KI¬
bov Ky 82 - - D | S - 1 - P y - - - - - -G ---DK.
sq Kv 82 -
00 ---v ---A--N-g---pK
rat Kv Bl VSEEGRKOONKLKDLSPIAERLGC
rat Kv 82 L - - ---R --A---E- 9A---- - - -
bov Kv 82 |  | - - - - - R -  |  | - - -E-Al- -- - -- -
sq Kv 82 L--D -- ---
REVAVVAIDE ---
rat Kv BI TLPOLAVAWCLRNEGVSSVLLGSS
rat Kv 82 - - - - -  | 1 | - - - - - —     A-
bov Kv ß2 - - - - - -  | - - - - - - — — — — — — — — -A
sq Kv ß2 s-A-- -I
----- - -T-HC -- -
a
rat Kv BI TPEOLIENLGAIQVLPKMTSHVVN
rat Kv B2 NA- --M--I- -------(s-SI-H
bov Kv 82 SAID  - M - - | 1 | - - ----LS-SIIH
sq Kv 82 slv p-- v --10---Yv---PTLM.
rat Kv BI EIDNILRNKPYSKKDRS
rat Ky 82 --- § --G- - - - - - - - -
bov Kv B2 - - - S - - Gl--- - - - - - - -
sq Kv B2 - L-KL-G---L-R--HPPRVFNNP..
2.tr. opt.
Appendix A
n.ret.1
n.ret.2
n.ret.3
ceam lar.
e.vis 2out.
out.
gaa.

4
T
plex.






8
—œamin.

grin.
S
f
ax.ter.

palis-

ce.vis.2.
ce palis
2.fro.
22


32










z.ra.



ce mult.lar.

ce mult lar.

ce multam



—ce.vis 3



5

ce mult lar.
-n.f.cent.
ce.vis.3
z.tan.
ax.ter.
cecent.



2











3
55
g

nfcent.
ce vis.3
ceen S

Olmm
n.f.cent.

ce.mult. lar.

from J. Z. Young, Central Nervou