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Volume 16, Number 15,
Issue of August 1, 1996
pp. 4799-4809
Copyright ©1996 Society for Neuroscience
Protein Kinase and G-Protein Regulation of Ca2+
Currents in Hermissenda Photoreceptors by 5-HT and GABA
Ebenezer N. Yamoah and
Terry Crow
Department of Neurobiology and Anatomy, University of Texas Medical
School, Houston, Texas 77225
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The effects of serotonin (5-HT) and GABA on two
Ca2+ currents, a transient low-voltage-activated
current (tLVA) and a sustained high-voltage-activated current (sHVA)
were examined in isolated photoreceptors of Hermissenda. The
sHVA current was blocked by 5-HT and reduced by activation of protein
kinase C (PKC) with phorbol 12-myristate 13-acetate. The effects of
5-HT were transiently reversed by staurosporine and partially blocked
by the PKC inhibitor peptide [PKC(19-36)]. GABA enhanced both the
tLVA and sHVA currents at low concentrations (5 nM to 5 µM) and reduced
the sHVA current at high concentrations (>10
µM). The GABA-mediated enhancement of the
Ca2+ current at low concentrations was sensitive
to block by picrotoxin. The protein kinase A (PKA) inhibitor peptide
[PKI(6-22)amide] blocked enhancement of both
Ca2+ currents produced by cAMP analogs and GABA,
suggesting that the effects at low concentrations may be PKA mediated.
Caged GTP- -S released by flash photolysis reduced the sHVA current,
and pretreatment of the photoreceptors with pertussis toxin blocked the
effects of higher concentrations of GABA, indicating that at higher
concentrations, the effects may be G-protein mediated.
Key words:
calcium current;
-aminobutyric acid;
neuromodulation;
cellular plasticity;
Hermissenda;
serotonin
INTRODUCTION
Neural networks can perform diverse functions by
the combined actions of classical transmitters and neuromodulators (for
review, see Harris-Warrick and Marder, 1991 ). Modulatory inputs may
produce diverse activity patterns of neural networks by activating
different second messengers that affect several membrane conductances.
The photoreceptors of Hermissenda crassicornis are sites not
only for the process of phototransduction but also for
Ca2+-dependent neuronal plasticity (Alkon and
Rasmusson, 1987; Crow, 1988). Numerous studies have provided evidence
that Ca2+, neurotransmitters/neuromodulators and
second messengers contribute to enhanced excitability of identified
type B photoreceptors observed after classical conditioning (Alkon,
1984; Farley and Auerbach, 1986; Crow et al., 1991; Falk-Vairant and
Crow, 1992; Matzel and Alkon, 1991; Matzel and Rogers, 1993).
Voltage-clamp studies of the B photoreceptors in conditioned animals
have revealed a reduction in the amplitude of two
K+ currents, the transient
(IA) and
Ca2+-activated K+
(IK,Ca) currents (Alkon et al., 1985;
Farley, 1988). Serotonin (5-HT) produces reductions of
IA and IK,Ca
that are similar to changes found in conditioned animals (Farley and
Wu, 1989; Acosta-Urquidi and Crow, 1993). The reduction of
IA and IK,Ca by
5-HT may be mediated by the activation of protein kinase C (PKC)
(Farley and Auerbach, 1986). In addition, GABA paired with
depolarization of the photoreceptors produces enhanced excitability of
the B photoreceptors (Matzel and Alkon, 1991; Alkon et al., 1993).
Although the mechanism for the contribution of GABA to enhanced
excitability in type B photoreceptors is unknown, recent evidence
suggests that GABA increases intracellular Ca2+
at the synaptic terminals of the photoreceptors (Alkon et al.,
1993).
To further examine modulation of membrane currents in
Hermissenda, we studied the effects of GABA and 5-HT on two
recently characterized Ca2+ currents in the type
A and type B photoreceptors, a transient low-voltage-activated (tLVA)
current and a sustained high-voltage-activated (sHVA) current (Yamoah
and Crow, 1994a), and provide evidence for the contribution of second
messengers to the modulation. At low concentrations ( 5
µM), GABA increased the tLVA and sHVA
Ca2+ currents, whereas at high concentrations
(>10 µM), GABA blocked the sHVA current. The
sHVA Ca2+ current was reduced by 5-HT. cAMP
analogs enhanced both the tLVA and sHVA Ca2+
currents. The effects of GABA and cAMP were blocked with inhibitors of
protein kinase A (PKA). High concentrations of GABA may produce effects
that are mediated by a G-protein, as shown by the reduction in the sHVA
current by release of caged GTP--S and pretreatment with pertussis
toxin (PTX). Activators of PKC reduced the sHVA
Ca2+ current; however, the time course of the
effect was delayed (>10 min) compared with the effects of 5-HT (<5
min) on Ca2+ currents. These results suggest that
the effect of 5-HT on Ca2+ currents may involve
PKC, a direct action on the channels, or an as yet unidentified second
messenger. A preliminary report of these results has been presented
(Yamoah and Crow, 1994b).
MATERIALS AND METHODS
Cell preparation. Adult Hermissenda were
obtained from Sea Life Supply (Sand City, CA). The photoreceptors were
isolated using the protocol outlined in Yamoah and Crow (1994a).
Briefly, the nervous systems were dissected from the animal and
incubated for 10 min at 4°C in 1.0 mg/ml protease type VIII
(Sigma, St. Louis, MO) and 7 mg/ml dispase grade II (Boehringer
Mannheim, Mannheim, Germany) in artificial seawater (ASW) composed of
(in mM): 420 NaCl, 10 KCl, 10 CaCl2, 22.9 MgCl2, 25.5 MgSO4, 15 HEPES (free acid) at pH of 7.8 (NaOH).
The initial enzyme treatment allows slow diffusion into the
interstitial space of the nervous system. The nervous systems were
transferred to room temperature (21°C) for 10 min and then
incubated at 4°C for 20-30 min in fresh ASW. This procedure not
only digested the connective tissue and glia around the photoreceptors,
but also loosened the capsule surrounding them. The eyes were pinched
off from the nervous system, desheathed in 35 mm sterile culture
dishes, and placed under an inverted microscope. Photoreceptors were
identified as type A or type B on the basis of their position relative
to the lens and the optic nerve. Isolated eyes without a lens and the
stump of the optic nerve were therefore discarded. Desheathed
photoreceptors were isolated from the eyes using mechanical agitation
and a fire-polished pipette. The average yield for this procedure was
two photoreceptors per eye. Isolated cells were incubated in ASW with
10 mM glucose and 50 mg/ml gentamicin sulfate
(Sigma) at 4°C before electrophysiological experiments were
performed.
Unless indicated, all chemicals were obtained from Sigma. A stock
solution of 5 mM GABA was made with bath solution
and stored at  20°C. Aliquots of the stock solution were added
to bath solutions to achieve a desired concentration (5 nM-1000 µM) and perfused
into the experimental chamber (0.8-1.0 ml) at a rate of 1.0-1.5
ml/min. A solution of 10 mM of
5-hydroxytryptamine creatinine sulfate complex (5-HT) was prepared and
applied as that described for GABA. Stocks solutions of 1 mM staurosporine (Calbiochem, La Jolla, CA), 5 mM isobutylmethylxanthine (IBMX) (Calbiochem),
membrane-permeable analogs of 5 mM cAMP,
8-(4-chlorophenylthio)-cAMP, and 5 mM
cAMP-N6,O2 -dibutyryl
(Calbiochem) were made in 100% dimethylsulfoxide (DMSO, Fisher
Scientific, Fair Lawn, NJ). These solutions were applied to the bath to
give final concentrations from the nanomolar to micromolar range,
depending on the agent. The final concentration of DMSO in the bath was
less than 0.2%. Stock solutions of synthetic peptide inhibitors of PKA
[PKAI(6-22)amide] and PKC [PKC(19-36)] (Gibco, Grand Island, NY)
were reconstituted in distilled water (500 µM)
and stored at 20°C. Aliquots of these solutions were added to
the pipette solution to make a final concentration of 5-20
µM in the peptide. All experiments with peptide
inhibitors were performed within 48 hr of reconstitution of the stock
solution. PTX and guanosine 5 -O-(2-thiodiphosphate)
trilithium salt (GDP- -S) and guanosine
5-O-(3-thiophosphate) tetralithium salt, (GTP--S) were
obtained from Sigma and Calbiochem. ``Caged'' GTP--S was obtained
from Molecular Probes (Eugene, OR).
Recording solutions. Bath solutions were made from (in
mM): 400 tetraethylammonium (TEA) acetate, 10 CaCl2, 50 MgS04, 5 4-aminopyridine (4-AP), 15 HEPES (free acid), pH 7.7, with
TEA-hydroxide and CsOH (TEA/OH). The pipette solution was made of (in
mM): 20 NaCl, 2 MgCl2, 10 EGTA, 20 TEACl, 300 CsCl, 300 N-methyl-D-glucamine (NMG), 10 glutathione (reduced), 5 Mg(ATP), 1 Na2(GTP),
40-50 HEPES, pH 7.4 (TEA-OH). Nystatin (0.1 µM) was prepared in 100% methanol. Osmolarity
of all solutions ranged from 0.96 to 1.00 Osm. The pipette solution was
stored in 15 ml aliquots at 20°C, and fresh pipette solutions
were used daily.
Voltage clamp. Ca2+ currents were
recorded with a standard patch-clamp technique at the whole-cell
configuration (Hamill et al., 1981) using the Axopatch 200A patch-clamp
amplifier (Axon Instruments, Foster City, CA). Patch pipettes were
pulled from borosilicate glass (World Precision Instruments, Sarasota,
FL) with a Flaming Brown micropipette puller, model P80/PC (Sutter
Instruments, San Rafael, CA). The tips of the pipettes were fire
polished and coated with SYLGARD #184 (Dow Corning, Midland, MI).
Pipettes had a final resistance of 0.7-1.6 M . Nystatin patches were
established by filling the tips of the pipettes with the nystatin
solution and backfilling with the pipette solution. Seals were
established by the application of negative pressure at the end of the
tubing connected to the pipette holder. Seal resistance ranged from 0.8 to 1.2 G. Series resistance was 3.3 ± 1.2 M
(n = 29) and 7.8 ± 2.6 M (n = 6) for nystatin patches. Series resistance was minimized with a
compensatory circuit. Current records were filtered at 5 kHz and
digitized at a frequency of 10 kHz with a Digidata interface (Axon
Instruments). Ca2+ currents were evoked with
command pulses from a personal computer (Gateway, Sioux City, SD) at a
sample rate of 250 µsec/point using PCLAMP software version 5.7.1 and
an A/D converter (Digidata, Axon Instruments). Passive leakage current
was subtracted on-line with a mean of four hyperpolarizing pulses one
fourth the size of the test potential. Photolysis was achieved by a
Chadwick Helmuth Strobex Model 278 xenon arc flash lamp (230 J maximum
output). The light was collected with an ellipsoidal mirror and the
spectrum reflected by a dichroic mirror (Acton Research, Acton, MA)
onto the aperture of a liquid light guide (Oriel, Stratford, CT).
Aperture output of the liquid light guide was focused onto the cell
using a plano-convex condenser lens and the microscope objective (Nikon
Fluor 20×, M.A. 0.4). Wavelength selection was enhanced between the
condenser and the preparation by reflecting the UV light off a second
dichroic mirror centered at ~350 mm. Electromagnetic
impulse-dependent current artifact lasted a few milliseconds.
Data analysis. Analysis of the current traces was made with
CLAMPFIT software (Axon Instruments). The criteria used for acceptance
of current records were similar to that outlined in Yamoah and Crow
(1994a). (1) Series resistance (except for nystatin-containing
electrodes) and seal resistance were <4.5 M and >0.6 G,
respectively. (2) Active current traces did not exhibit ``pumps.''
(3) The current-voltage relation had a negative peak as the step
potentials approached the apparent reversal potential of
Ca2+. Statistics were computed with SigmaPlot
software (Jandel Scientific, San Rafael, CA). Descriptive data are
presented as means ± SD. Statistical differences within groups
were determined using t tests for correlated means, and
between-group differences consisted of t tests for
independent groups. Two-tailed tests were used in the statistical
analysis unless otherwise indicated.
RESULTS
Effect of 5-HT on ICa
Calcium currents and their modulation by 5-HT and GABA were
studied in both type A and type B photoreceptors by blocking the
outward currents IA,
IK,V, and IK,Ca
with 4-AP, TEA, and Cs+. Inward
Na+ current was blocked by choline substitution
of Na+, and the inward rectifier current was
blocked by Cs+. Two components of
Ca2+ currents, a tLVA and an sHVA, have been
identified previously in the photoreceptors (Yamoah and Crow, 1994a,b).
Type A and type B photoreceptors were identified on the basis of their
anatomical positions in the eyes. Isolated cells near the lens were
classified as type A, and cells in the posterior part of the eye near
the stump of the optic nerve were classified as type B. Further
classification of A and B photoreceptors as medial or lateral was not
possible with these isolation procedures. The mean peak
Ca2+ current recorded from type A and type B
photoreceptors was not statistically different (type A:
 = 1.1 ± 0.41 nA, n = 16;
type B: = 1.2 ± 0.41 nA, n = 15; t29 = 0.66, NS). The sHVA
Ca2+ current recorded from both type A and type B
photoreceptors was reduced by the application of 5-HT. An example of
the tLVA and sHVA Ca2+ currents recorded from a
type A cell is shown in Figure 1A.
The whole-cell currents were elicited from a holding potential of 80
mV. With EGTA, ATP, and GTP in the patch pipette, the
Ca2+ currents remained stable for at least 60 min
after establishing the whole-cell configuration. As shown in Figure
1B, the application of 25 µM 5-HT
blocked the sHVA current and had little, if any, effect on the tLVA
current (see Fig. 1C). The magnitude of the reduction in the
sHVA current in both type A and type B cells by 5-HT was similar (type
A control: = 1.1 ± 0.4 nA; 5-HT:
= 0.59 ± 0.3 nA; n = 15;
t14 = 8.8; p < 0.0001)
(type B control: = 1.2 ± 0.4 nA; 5-HT:
= 0.65 ± 0.3 nA; n = 15;
t14 = 10.9; p < 0.0001).
The time course of the effect of 5-HT on the sHVA current (<5 min) was
faster than the time course of current rundown (>60 min). The
current-voltage relationship shown in Figure 1C revealed
two apparent peaks (~5 and 30 mV) measured under control conditions
(open circles) before the application of 5-HT. However, in
the presence of 25 µM 5-HT, only one peak was
expressed at 5-10 mV (filled circles, Fig. 1C).
At holding potentials greater than or equal to 30 mV, at which the
sHVA Ca2+ current was predominantly activated,
5-HT reduced the magnitude of the current without altering the
inactivation kinetics. The effects of 5-HT were reversible within 5-7
min after washout (see Fig. 2C). Figure 1D
illustrates the dose-response relationship for the effects of 5-HT on
the sHVA Ca2+ current. The half-blocking
concentration of 5-HT on the sHVA Ca2+ current
was 2.4 µM. The concentrations of 5-HT applied
to isolated photoreceptors were lower than that reported previously for
isolated nervous systems (Farley and Wu, 1989). Presumably higher
concentrations are required for intact isolated nervous systems to
overcome diffusional barriers and possible 5-HT uptake by both neurons
and glial cells.
Fig. 1.
Effect of 5-HT on the sHVA
Ca2+ current. Traces were recorded from a type A
cell. Superimposed Ca2+ currents in A
and B were elicited from a holding potential of 80
mV to step depolarizations of 5 and 28 mV. A, Two
Ca2+ currents were recorded: a tLVA current and
an sHVA current. B, The sHVA
Ca2+ current was blocked by 5-HT (25 µM). The inset consists of
superimposed current traces elicited at 5 mV for control and after
the application of 5-HT (25 µM). The difference
current that represents the current blocked by 5-HT is shown by the
dotted line. The current that was blocked by 5-HT was
predominantly the sustained component of the Ca2+
current. C, Current-voltage plot of
Ca2+ current before (open circles)
(n = 7; data were from 3 type A and 4 type B
cells) and after the application of 5-HT (filled
circles) (n = 7). Currents were corrected for
passive leakage current, and the peak current was plotted for each step
potential. The control plot had two apparent peaks (open
arrows). The application of 5-HT (25 µM) reduced the current, resulting in an
I-V plot showing a single peak (closed
arrow). D, Dose-response relationship. Normalized
current was plotted for different 5-HT concentrations, and a curve
through the points was fitted with a logistic function
(n = 5). Normalized current used to generate the
dose-response curve followed the equation (I Imin)/(Imax Imin), where I is the current
magnitude at a given concentration of 5-HT and
Imin is the maximum current at which the
effect of the 5-HT had saturated. The saturation concentration for
5-HT, GABA, and baclofen was 1 mM, and 2 µM for phorbol esters (see Figs. 2, 3, 4).
Imax is the maximum control current before
the application of 5-HT. The estimated half-blocking concentration of
5-HT was 2.4 µM.
[View Larger Version of this Image (20K GIF file)]
Fig. 2.
Effects of PMA and inhibitors of PKC on
Ca2+ currents. A, Mean dose-response
curve showing the relationship between PMA concentration and reduced
sHVA Ca2+ current. The estimated half-blocking
concentration of PMA was 12.3 nM with a power of
2 from the logistic function used to fit the data points. Data points
were generated from four different experiments from two type A and two
type B cells. B, Control traces (type A cell) were elicited
from a holding potential of 80 mV to a step potential of 20 mV in
normal bath solutions and in a solution containing PMA (20 nM) (B1) or 4 -phorbol (50 nM) (B2) (type A cell). As shown in
B1, PMA (20 nM) reduced, but did not
eliminate, the sHVA Ca2+ current, and
4-phorbol did not affect the sHVA current (B2).
B3, Ca2+ currents, from a type B cell,
were recorded in the presence of the PKC inhibitor (5 µM) [PKC(19-36)] (PKCI).
In the presence of PKC(19-36), 5-HT (50 µM)
had a small effect on the Ca2+ current.
B4, The broad-spectrum kinase inhibitor staurosporine (20 nM) transiently (3-5 min) reversed the effects
of 5-HT (traces were recorded from a type B cell). However, 10 min
after the application of staurosporine in the presence of 5-HT, the
sHVA Ca2+ current was reduced to the original
5-HT-induced magnitude. C, Time course of 5-HT and PMA
effects on Ca2+ currents. The photoreceptor was
held at 80 mV and stepped to 20 mV to activate the sHVA component of
the Ca2+ current. The magnitude of the current
after application of 5-HT (20 µM) and after
washout is shown. 5-HT blocked the sustained current elicited at 20 mV
(filled circles). PMA (20 nM)
reduced the sHVA Ca2+current
(filled boxes); however, the time course of action
was delayed compared with the effects of 5-HT.
[View Larger Version of this Image (21K GIF file)]
Effects of PKC activators and inhibitors on
Ca2+ current
Previous research has shown that the effects of 5-HT on diverse
K+ currents may be mediated by PKC (Farley and
Auerbach, 1986; Crow et al., 1991). A potential role of PKC in
mediating the effects of 5-HT on the sHVA Ca2+
current was examined by measuring currents after the application of the
PKC activator phorbol 12-myristate 13-acetate (PMA). The dose-response
curve is shown in Figure 2A. The
phorbol ester PMA is an effective activator of PKC in
Hermissenda photoreceptors (Crow et al., 1991). Whereas 5-HT
completely blocked the sHVA current, PMA partially reduced the current
as shown in Figure 2B1. The statistical analysis of the
difference scores for the 5-HT and PMA group data showed that 5-HT
resulted in a significantly larger reduction in the sHVA current
compared with PMA (5-HT:  = 0.78 ± 0.05 nA;
n = 9; PMA: = 0.40 ± 0.08 nA;
n = 9, t16 = 4.11, p < 0.005). The inactive analog of phorbol ester, 4 -phorbol, did not affect either component of the
Ca2+ current as shown in Figure 2B2.
It is possible that PMA not only reduced sHVA but also removed the
inactivation of the transient component of the
Ca2+ current. However, this is unlikely, because
PMA did not affect currents from cells that predominantly expressed the
tLVA current (data not shown). Thus, the effects of PMA cannot be
attributed to its secondary effect on either the changes in the
magnitude or the kinetics of the tLVA current.
The phorbol ester produced different kinetic effects on the sHVA
current compared with 5-HT. However, this finding may be the result of
the effect of phorbol esters on other cellular processes in addition to
activation of PKC. A specific inhibitor of PKC (PKCI), [PKC(19-36)],
reduced but did not completely block the effects of 5-HT on the sHVA
current (Fig. 2B3). In addition, staurosporine, a broad
spectrum kinase inhibitor (Tamaoki et al., 1986; Yanagihara et al.,
1991) transiently (3-5 min) reversed the effects of 5-HT (note that
5-HT was applied 3 min before staurosporine was perfused into the bath)
(see Fig. 2B4). However, 10 min after application of both
5-HT and staurosporine, the Ca2+ current was
reduced. It is conceivable that activation of PKC by 5-HT might
increase over time and that prolonged 5-HT exposure may overcome the
inhibitory effects of staurosporine.
We observed that the reduction in the Ca2+
current produced by 20 nM PMA was delayed
relative to the effects of 20 µM 5-HT, as shown
in Figure 2C. The effects of 5-HT on sHVA currents were
observed within 3 to 5 min of application; however, the effects of PMA
were not observed until 10-15 min after bath application. A summary of
the group data and statistical results for the effects of 5-HT, active
and inactive phorbol esters, and PKC inhibitors is presented in Table
1.
Effect of GABA on Ca2+ current
We investigated the effects of GABA on the two voltage-activated
Ca2+ currents by using known analogs and
inhibitors of GABA receptors. The effect of GABA on the sHVA current
was studied from a holding potential greater than 35 mV at which the
tLVA current is predominantly inactivated. At low concentrations
(5-1000 nM), GABA enhanced the sHVA current in
both type A and B photoreceptors (type A control: = 1.18 ± 0.4 nA, 15 nM; GABA:
= 1.43 ± 0.38 nA; n = 6, t5 = 6.08, p < 0.005)
(type B control: = 1.13 ± 0.47 nA, 15 nM; GABA: = 1.43 ± 0.45 nA; n = 7, t6 = 7.66, p < 0.005). The data were obtained from the peak
current elicited at 20 mV. An example from a type B cell of enhancement
of sHVA by 10 nM GABA is shown in Figure
3, A and A2. In contrast to the
enhancement of sHVA by l0 nM GABA, 30 µM GABA reduced the sHVA current as shown by
the example in Figure 3A3. At higher concentrations
(~10-1000 µM), GABA reduced the sHVA current
compared with the controls in both type A and B cells (type A control:
= 1.17 ± 0.32 nA, 25 µM; GABA: = 0.66 ± 0.20 nA; n = 6, t5 = 8.98, p < 0.005) (type B control: = 1.12 ± 0.31 nA, 25 µM; GABA:
= 0.72 ± 0.21 nA; n = 7, t6 = 5.52, p < 0.005).
However, we did not qualitatively detect a reduction of the tLVA
current in the presence of high concentrations of GABA at step
potentials at which we assumed that the tLVA current was activated. A
current-voltage plot for a control and two concentrations of GABA (10 nM and 30 µM) is shown in
Figure 3B. A dose-response curve illustrating the effects
of low concentrations of GABA on sHVA is shown in Figure 3C.
At holding potentials at which the tLVA Ca2+
current can be activated (less than or equal to 50 mV), enhancement
of the current by low concentrations of GABA was also observed (see
difference current from a type A cell in Fig. 3D). The
effects of low concentrations of GABA at holding potentials at which
the tLVA Ca2+ can be activated was similar for
both type A and type B cells (type A control: = 0.19 ± 0.08 nA, 15 nM; GABA:
= 0.24 ± 0.08 nA; n = 7, t6 = 9.95, p < 0.005)
(type B control: = 0.17 ± 0.07 nA, 15 nM; GABA: = 0.22 ± 0.07 nA; n = 9, t8 = 9.05, p < 0.005).
Fig. 3.
Effects of GABA on Ca2+
current. A, A family of superimposed
Ca2+ currents elicited from a type B cell at a
holding potential of 30 mV. A1, Control; A2,
after the application of 10 nM GABA;
A3, after the application of GABA (30 µM). B, Current-voltage plot of
control and GABA-induced effects on the Ca2+
current. Graph was generated from current traces shown in
A1-A3. From a holding potential of 30 mV, the
sHVA Ca2+ current was the predominant
Ca2+ current activated by the command steps. A
low concentration of GABA (10 nM) enhanced the
current. Higher concentrations of GABA (30 µM)
reduced the sHVA Ca2+ current. C,
Dose-response curve was generated using the same equation as described
in Figure 1D. The half-activation concentration for GABA at
low concentrations as estimated from four experimental data points with
a logistic function was 12.5 nM using a power of
1.5 as the best fit. Imax was equal to the
current recorded at the saturation concentration of GABA between 500 and 1000 nM. For each cell, a profile of the
effect of GABA from a lower to higher concentration was plotted as the
example shown in Figure 4E. Imax
was the peak of the profile of the biphasic concentration curve.
Imin is the current measured before GABA
application. D, At low concentrations, GABA enhanced the
tLVA Ca2+ current (see A). Traces from
a type A cell were generated from a holding potential of 80 mV to a
step depolarization of 10 mV. The trace in a is in the
presence of 10 nM GABA, and the trace in
b is the control. The difference current (dotted
line) corresponds to the increase in the tLVA
Ca2+ current attributable to GABA.
[View Larger Version of this Image (22K GIF file)]
The two effects of GABA were studied further with baclofen, a
GABAB receptor agonist. In preparations perfused
with 25 µM baclofen, there was a steady-state
reduction in the Ca2+ currents compared with
control current measurements (see Table 2). Figure
4B shows an example of the steady-state
effects of baclofen compared with control conditions in Figure
4A. These results suggest that activation of
GABAB receptors is sufficient to produce
inhibition of Ca2+ currents, an observation
similar to that reported from studies of dorsal root ganglion (DRG)
neurons (Scott et al., 1990). It has been postulated that two subtypes
of GABAB receptors are present in the
photoreceptors, one gating K+ channels and
another regulating closure of K+ channels (Alkon
et al., 1993). However, after enhancement of the
Ca2+ current with GABA (50 nM), the application of picrotoxin (1 µM), a GABAA receptor
antagonist, inhibited the current (Fig. 4C). These results
suggest the possible involvement of either GABAA
receptors or a subclass of as yet unidentified GABA receptors in the
enhancement of Ca2+ currents. Dose-response
curves for baclofen and a high concentration of GABA are shown in
Figure 4D. The concentration profile of the effects of GABA
on the sHVA Ca2+ current is plotted in Figure
4E.
Table 2.
Effects of GABA, baclofen, picrotoxin, cAMP analog, IBMX,
PKAI, and PKCI on calcium currents
Control
|
Experimental
|
df |
t |
p
|
| Treatment |
nA |
Treatment |
nA |
|
 |
0.95
± 0.29 (7) |
GABA* (10 nM) |
1.09 ± 0.38
(7) |
6 |
2.5 |
<0.05 |
| |
0.17 ± 0.06
(7) |
GABAI,t (10 nM) |
0.23 ± 0.04
(7) |
6 |
3.1 |
<0.025 |
| |
0.94 ± 0.30 (7) |
GABA*
(30 µM) |
0.67 ± 0.20 (7) |
6 |
3.0 |
<0.025
|
| |
1.01 ± 0.11 (9) |
Baclofen (25 µM) |
0.66 ± 0.27 (9) |
8 |
3.4 |
<0.01
|
| GABA (50 nM) |
0.78 ± 0.21 (6) |
GABA (50 nM) + Picro (1 µM) |
0.19 ± 0.16
(6) |
5 |
9.5 |
<0.005 |
| |
1.28 ± 0.16
(7) |
Dibu.cAMP (2.5 nM) |
2.17 ± 0.33
(7) |
6 |
7.5 |
<0.005 |
| |
0.90 ± 0.18 (5) |
IBMX
(200 µM) |
1.16 ± 0.21
(5) |
4 |
4.3 |
<0.025 |
| PKAI (5 µM) |
1.00
± 0.21 (5) |
PKAI (5 µM) + GABA (50 nM) |
0.43 ± 0.13 (5) |
4 |
4.8 |
<0.01
|
| PKCI (5 µM) |
1.08 ± 0.26 (6) |
PKCI (5 µM) + GABA (25 µM) |
0.57 ± 0.12
(6) |
5 |
3.7 |
<0.025 |
|
|
Asterisk indicates that the Vh was 30 mV
and step potential was +20 mV. Vh for other
experiments was 80 mV, and the step voltage was 20 mV. The
transient component of the calcium current (It)
was elicited from a holding potential of 80 mV to step potentials of
10 mV. Data are expressed as the mean peak current ± SD (number
of cells). Computed t statistics are for tests of correlated
means. Picro, Picrotoxin; dibu.cAMP,
cAMP-N6,O6-dibutyril.
|
|
Fig. 4.
Effects of a GABAB agonist
and a GABAA antagonist on
Ca2+ currents. A, Control traces
generated from a holding voltage of 80 mV to step voltages of 15
and 20 mV. B, Baclofen (25 µM)
reduced the currents compared with the control. C, In a
different cell, a control current trace was elicited by stepping from
80 to 20 mV followed by the bath application of GABA (50 nM). The current was enhanced in the presence of
GABA. The application of the GABAA antagonist
picrotoxin (1 µM) reduced the current.
D, Dose-response curve for high concentrations of GABA and
baclofen. Curves were generated as described in the legends for Figures
1, 2, 3. Because GABA elicited a dual effect, a concentration profile of
the effects of GABA on each cell was generated as shown in
E. Normalized current used to generate the dose-response
curve followed the equation (I Imin)/(Imax Imin), where I is the current
magnitude at a given concentration of GABA and
Imin is the minimum current at which the
effect of GABA had saturated (1 mM).
Imax is the plateau current extrapolated
from E. The half-blocking concentrations for GABA and
baclofen estimated from these curves were 62.5 and 25.5 µM, respectively. E, Graph of the
concentration profile of GABA on the sustained
Ca2+ current. The effect of GABA on the
Ca2+ current shows an initial rise in the current
magnitude at low concentrations and drop in the size of the current as
the concentration of GABA increases. The peak current was observed at
~100 nM GABA.
[View Larger Version of this Image (19K GIF file)]
Effects of cAMP on calcium current
Administration of dibutyryl-cAMP or chlorophenylthio-cAMP (2.5 mM) produced an increase in the amplitude of the
Ca2+ current at both low- and high-step voltages
from negative holding potentials (less than or equal to 50 mV) (see
Fig. 5A1, A2). The profile of the
cAMP analog-induced current at 10 and 20 mV step depolarizations
suggests that the current consisted of both the tLVA and sHVA
Ca2+ currents. The analysis of the group data
elicited by a voltage step to 10 mV from a holding potential of 80
mV revealed that the cAMP analogs resulted in a significant enhancement
of the tLVA current (control: = 0.27 ± 0.05 nA; cAMP: = 0.44 ± 0.09 nA; n = 4, t3 = 3.79, p < 0.05).
The cAMP analog-induced current exhibited inactivation. At holding
potentials at which the sHVA Ca2+ current was
predominantly activated (greater than or equal to 30 mV), a similar
enhancement of the Ca2+ current was observed in
the presence of the cAMP analogs. The effects of IBMX, a
phosphodiesterase inhibitor known to enhance the actions of
cAMP-dependent phosphorylation, suggest that the increase in the
Ca2+ current was mediated by PKA (see Fig.
5B1, B2, Table 2). In the presence of IBMX, the
tLVA Ca2+ current elicited by a voltage step to
10 from 80 mV was enhanced (control: = 0.25 ± 0.08 nA, IMBX: = 0.41 ± 0.13 nA;
n = 3, t2 = 4.56, p < 0.05).
Fig. 5.
Effects of a cAMP analog, IBMX, and PKAI on
Ca2+ currents. Current traces were generated
before and after the bath application of cAMP analogs (2.5 mM); both the dibutyryl and the chlorophenylthio
analogs of cAMP had similar effects. The difference current traces
after the treatment are represented by the dotted lines. The
cAMP analogs increased both tLVA (A1) and sHVA
(A2) Ca2+ currents. Similar results
were obtained after the application of IBMX (200 µM), as shown in B1 and
B2. After dialysis of cells with PKAI (5 µM), GABA (50 nM) reduced
the magnitude of the Ca2+ currents as shown in
C1 and C2. Traces in A and
B were generated from type A cells, and traces in
C were from a type B cell. D, Group data showing
the effects of both cAMP analogs (n = 7) and IBMX
(n = 5) on the sHVA Ca2+ current
and the effect of GABA on the sHVA Ca2+ current
in the presence of PKAI (n = 6).
[View Larger Version of this Image (21K GIF file)]
To test whether the effects of low concentrations of GABA (see Fig.
3A) were mediated by PKA, cells were first perfused
internally with the PKA inhibitor (PKAI), [PKI(6-22)amide] (5 µM) before the bath application of GABA. After
dialysis of cells with PKAI, application of GABA (50 nM) reduced the Ca2+
current as shown in the example in Figure 5C. The group data
for the effects of the cAMP analogs, IBMX, and PKAI on the sHVA current
are shown in Figure 5D, and the statistical analysis of the
group data is included in Table 2.
Possible [PKC(19-36)] effects on the actions of GABA
As shown in Figures 2B and 3A, PMA and GABA,
at micromolar concentrations, reduced the magnitude of the sHVA
Ca2+ current. The effects of PMA on the current
waveform and time course exhibited different kinetics from the effects
of GABA. To further investigate a possible relationship between GABA
and PKC we examined the effects of GABA on the
Ca2+ currents in the presence of the PKC
inhibitor PKCI [PKC(19-36)]. In the presence of PKCI (5 µM) in the patch pipette solution, micromolar
concentrations of bath-applied GABA (25 µM)
reduced the sHVA current. Thus, PKC does not appear to contribute to
the modulation of Ca2+ currents by GABA. The
results of the statistical tests of data generated from various
treatments are summarized in Table 2.
G-protein-mediated reduction of Ca2+ current
Previous research has implicated G-proteins as a target for
Ca2+- mediated plasticity in the photoreceptors
(Matzel and Alkon, 1991). We examined this further by studying the
effects of G-proteins on Ca2+ currents to
determine which second messengers are responsible for the GABA-induced
effects. As shown in the current-voltage plot in Figure
6A, the nonhydrolyzable form of GTP
(GTP--S) reduced the sHVA Ca2+ current
compared with normal controls or controls that were dialyzed with
GDP--S. The analysis of the group data revealed a statistically
significant reduction in Ca2+ currents after the
application of GTP--S (control: = 2.5 ± 0.24 nA; GTP--S: = 1.5 ± 0.11 nA;
n = 3, t2 = 3.15, p < 0.05, one-tailed test). In contrast, GDP--S did
not produce a significant reduction in the Ca2+
current (control: = 2.7 ± 0.18 nA;
GDP--S: = 2.23 ± 0.33 nA;
n = 2, t1 = 2.9, NS,
one-tailed test). We further confirmed the results by using a
photolabile form of GTP--S. Release of caged GTP--S caused a
decline in the magnitude of the Ca2+ current as
shown in Figure 6B1. UV light alone did not affect the
currents as shown in Figure 6B2. In cells treated with the
G-protein inhibitor PTX, the differential effects of GABA on
Ca2+ currents were abolished. After treatment
with PTX, high concentrations of GABA enhanced the
Ca2+ current as shown in Figure 6C.
The analysis of group data revealed that GABA (30 µM) enhanced Ca2+
currents if cells were pretreated with PTX (PTX control:
= 8 ± 0.08 nA, 30 µM; GABA: = 1.44 ± 0.09 nA; t2 = 43.9, p < 0.005). These results suggest that GABA may be acting through
at least two second messengers, G-proteins and PKA.
Fig. 6.
G-protein mediates GABA-induced reduction of
Ca2+ current. A, Current-voltage
relationship for data obtained from control experiments (open
circles), controls dialyzed with GDP--S (filled
boxes), and experiments performed in the presence of GTP--S (5 mM) (filled circles) in the
pipette. The magnitude of the sHVA Ca2+ current
was significantly reduced in the presence of GTP--S. Currents were
generated from 80 mV to a step potential of 30 mV. In contrast to the
effects of GTP--S, GDP--S (5 mM) failed to
reduce the current. Currents were generated from 80 mV to a step
potential of 30 mV. When GTP--S was released by flash photolysis
from caged GTP--S, the current was reduced as shown in a
representative trace in B1. Control recording in
B2 shows that the light flash does not reduce the
Ca2+ current. C, The effect of PTX on
Ca2+ currents. Photoreceptors were incubated in
PTX (1 µg/ml) for 6 hr followed by Ca2+ current
measurement, before (Control) and after applying GABA
(30 µM). The typical GABA-induced reduction of
the Ca2+ current was abolished by pretreatment
with PTX. In the presence of PTX, GABA only enhanced the
Ca2+ current. Currents were generated from 80
mV to a 10 mV step potential. All experiments in this section were
conducted in the presence of bath Ca2+ (20 mM).
[View Larger Version of this Image (18K GIF file)]
DISCUSSION
These results show that both 5-HT and GABA modulate
Ca2+ currents in the photoreceptors of
Hermissenda. 5-HT blocks the sHVA Ca2+
current; GABA increases both the tLVA and sHVA currents at low
concentrations, and blocks only the sHVA current at higher
concentrations. Studies of the photoreceptors in Hermissenda
have suggested that the mechanism of plasticity is
Ca2+-dependent (Falk-Vairant and Crow, 1992;
Matzel and Rogers, 1993). However, the mechanism of the induction and
maintenance of Ca2+-dependent neuronal plasticity
is not understood. Previous studies have implicated GABA in the
mediation of increases in intracellular Ca2+ at
the synaptic terminals of the photoreceptors (Alkon et al., 1992). In
addition, 5-HT produces enhanced excitability of type B photoreceptors
and synaptic facilitation of inhibitory synaptic connections between
type B and type A photoreceptors (Farley and Wu, 1989; Crow and
Forrester, 1991; Schuman and Clark, 1994). Recently, it was reported
that GABA, which under normal conditions is inhibitory, may transform a
synapse into an excitatory one by an unknown mechanism (Alkon et al.,
1992).
5-HT modulation of Ca2+ current
Modulation of Ca2+ currents by 5-HT has been
demonstrated in several neural systems. 5-HT reduces a
high-voltage-activated Ca2+ current in dorsal
raphe neurons (Penington and Kelly, 1990; Penington et al., 1992),
embryonic chick sensory neurons (Dunlap and Fischbach, 1981), and
spinal cord neurons (Sah, 1990). As a result of
Ca2+ current inhibition, neuronal excitability is
reduced (Penington et al., 1992) and action potential durations are
shortened (Dunlap and Fischbach, 1981; Penington et al., 1992). In
contrast, a low-voltage-activated Ca2+ current in
spinal motor neurons is enhanced by 5-HT (Berger and Takahashi, 1990).
Calcium currents in Helix aspera neurons (Paupardin-Tritsch
et al., 1986) and Aplysia sensory neurons are enhanced by
5-HT through activation of PKC (Edmonds et al., 1990; Braha et al.,
1993). However, in neuron R15 of Aplysia, potentiation of
Ca2+ currents by 5-HT is mediated by activation
of PKA (Levitan and Levitan, 1988). Previous work in
Hermissenda suggested that 5-HT increased
Ca2+ currents in type B photoreceptors (Farley
and Wu, 1989) and pedal neurons (Jacklet and Acousta-Urquidi, 1985).
The initial studies of Ba2+ currents in the type
B photoreceptors did not identify the two components of
Ca2+ currents that were recently characterized by
Yamoah and Crow (1994a). The failure to isolate these currents in
earlier studies may be attributable to differences in the experimental
procedures and potential current contamination in the voltage-clamp
data. Previous studies have reported that in type B photoreceptors,
5-HT reduced IA and
IK,Ca (Farley and Wu, 1989), slowed the
rate of inactivation of IK,V, and in some
cases reduced IK,V (Acosta-Urquidi and
Crow, 1993). Moreover, recent evidence suggests that the reduction of
IK,Ca by 5-HT is a consequence of
modulation of ICa by 5-HT (Yamoah and Crow,
1995). In addition, 5-HT enhances an inward rectifier current
(IIR) (Acosta-Urquidi and Crow, 1993),
which is conducted by Na+ and K+ (Matzel et
al., 1995). Voltage-clamp protocols used in earlier studies to measure
Ba2+ currents in the photoreceptors may not have
eliminated other potential ionic current contamination (Farley and Wu,
1989). Because some of these currents are not exclusively carried by
K+, shifting EK to 0 mV does not necessarily indicate that a null potential for these
conductances will be attained. If inward Ca2+
currents are recorded in the presence of any 5-HT-sensitive inward or
outward currents, the reduction of outward K+
currents or enhancement of the inward rectifier current by 5-HT may be
manifested in an apparent increase in a putative
Ca2+ current.
Previously, it was reported that 5-HT increased the amplitude of the
plateau phase of the generator potential (Farley and Wu, 1989; Crow and
Forrester, 1991) by reducing IK,Ca and
IA (Farley and Wu, 1989; Acosta-Urquidi and
Crow, 1993). In addition, 5-HT enhances the inward rectifier and shifts
the current's apparent reversal potential to more positive potentials
(Acosta-Urquidi and Crow, 1993). Such effects would be expected to
enhance the amplitude of the generator potential and potentially
prolong the afterdepolarization phase of light-elicited generator
potentials. Under such conditions, Ca2+ influx
should increase. However, a reduction of the sHVA
Ca2+ current by 5-HT may be a feedback mechanism
to prevent intracellular Ca2+ overload.
The reduction of IK,Ca and
IA by 5-HT has been attributed to
activation of PKC, because application of phorbol esters and
microinjection of PKC into the B photoreceptors produced similar
effects (Farley and Auerbach, 1986). The following evidence implicates
PKC as a possible candidate for 5-HT-mediated reduction of the sHVA
Ca2+ current. PMA, but not 4-phorbol, reduced
the Ca2+ current, and staurosporine transiently
(3-5 min) reversed the effects of 5-HT. PKCI reduced the effect of
5-HT on the sHVA Ca2+ current. However, the time
course of PMA reduction of the Ca2+ current in
the photoreceptors was delayed relative to the effects of 5-HT. Similar
delayed effects of phorbol esters have been demonstrated for changes of
action potential amplitudes in Aplysia bag cells (Conn et
al., 1989). In Aplysia sensory neurons, it has been reported
that the time course of the effect of 5-HT lags behind the time course
of the effects of phorbol ester (Braha et al., 1993). In this study, we
found that effects of 5-HT on Ca2+ current was
expressed earlier than the effect of PMA on the
Ca2+ current. However, the sequences of
activation of PKC by phorbol ester and transmitters are quite
different, because phorbol esters must permeate through the cell
membrane to activate PKC and, thus, the time courses of activation of
their effects are not expected to be identical. Taken together, these
results suggest that the reduction of the Ca2+
currents in the photoreceptors by 5-HT may be PKC mediated. In addition
to PKC modulation, a possible role of CAM kinase II in the modulation
of the Ca2+ currents has been suggested and
requires further investigation (Matzel and Alkon, 1991).
GABA modulation of Ca2+ currents
GABA has two effects on the amplitude of
Ca2+ currents in the photoreceptors that are
similar to effects reported for DRG neurons (Scott et al., 1990, 1991).
At nanomolar and micromolar concentrations, GABA increases and
decreases the Ca2+ currents, respectively.
GABA increases intracellular Ca2+ at the
axonal terminal of the photoreceptors as measured with
Ca2+ indicator dyes (Alkon et al., 1993).
Recently, it was proposed that the GABA-induced rise in intracellular
Ca2+ may be the trigger for the induction of
cellular plasticity (Alkon et al., 1993). In an independent study,
Matzel and Rogers (1993) observed that Ca2+ is
required for the induction of plasticity in the photoreceptors.
Although the intracellular Ca2+ release
hypothesis is intriguing, we propose that GABA-induced increases in
Ca2+ influx through voltage-activated
Ca2+ channels may augment the intracellular
release process, such as a Ca2+-induced
Ca2+ release. The contribution of these two
processes acting together may exceed a threshold for the induction of
Ca2+-dependent changes in the photoreceptors. It is
conceivable that beyond the threshold and the capacity of intracellular
Ca2+ buffers, reduction of the
Ca2+ currents by GABA may serve an important
physiological function by preventing possible cell death as a result of
intracellular Ca2+ overload.
What GABA receptors are responsible for the modulation of
Ca2+ current?
The finding that baclofen reduced the
Ca2+ currents suggests that a
GABAB receptor is involved in the modulation of
Ca2+ channels. Muzzio et al. (1994) have reported
similar reduction of Ca2+ currents by baclofen in
the photoreceptors. This is similar to the GABAB
receptor-mediated inhibition of Ca2+ currents in
hippocampal neurons (Pfrieger et al., 1994), cerebellar Purkinje
neurons (Mintz and Bean, 1993), and sensory neurons (Scott et al.,
1991). However, blockage of current enhancement by picrotoxin, a known
GABAA receptor antagonist (Puia et al., 1990),
suggests that there may be different subtypes of GABA receptors that
are sensitive to picrotoxin, but not similar to the known
GABAA anion receptor. The GABA receptor mediating
Ca2+ current enhancement may be a subtype of a
GABA receptor with some homologies to the GABAA
receptor, because it is known that different subunits of GABA receptors
can form receptors with different pharmacologies (Verdoorn et al.,
1990; Shimada et al., 1992). The possibility that a novel GABA receptor
(GABAC), which has been characterized in bipolar
neurons in salamander retina (Lukasiewicz and Werblin, 1994), is also
present in Hermissenda photoreceptors requires further
investigation. Alternatively, Alkon et al. (1992) have suggested the
presence of two subtypes of GABAB receptors that
have differential effects on membrane conductances in the
photoreceptors. In either case, the GABA enhancement of the
Ca2+ currents appears to be mediated through
activation of PKA, because cAMP and IBMX essentially mimic the GABA
effects, and PKAI blocked the GABA-induced enhancement of the
Ca2+ currents.
G-protein and GABA reduction of Ca2+ current
GABA-mediated reduction of the sHVA Ca2+
current may result from activation of a G-protein. The evidence for
this is as follows: GTP--S, but not GDP--S, reduced the sHVA
Ca2+ current. Micromolar concentrations of GABA
and baclofen also reduced the current. PKCI had no effect on the
actions of GABA. An inhibitor of some G-proteins (PTX) blocked the
reduction in the sHVA current typically produced by GABA at micromolar
concentrations. In PTX-treated cells, GABA enhanced the sHVA
Ca2+ current both at nanomolar and micromolar
concentrations. Thus, the dual effects of GABA on the
Ca2+ current is mediated through activation of
PKA and a G-protein.
Several examples of G-protein modulation of
Ca2+ currents have been reported from different
systems, e.g., G-protein augmentation of agonist and
antagonist actions of the dihydropyridine-sensitive
high-voltage-activated Ca2+ currents in sensory
and sympathetic neurons (Dolphin and Scott, 1989). In sympathetic
neurons, it has been suggested that G-proteins regulate
Ca2+ channels tonically (Scott and Dolphin,
1990). Interestingly, in DRG neurons, GABAB
receptors have been found to be coupled to Ca2+
channels via a G-protein (Scott et al., 1990). Moreover, GABA
has a dual effect on a low-voltage-activated Ca2+
channel in DRG neurons. In contrast, the dual effects of GABA seen in
Hermissenda photoreceptors were observed on the sHVA
Ca2+ current, but not the tLVA
Ca2+ current.
FOOTNOTES
Received Feb. 21, 1996; revised April 30, 1996; accepted May 3, 1996.
This work was supported by National Institutes of Mental Health Grant
MH40860 to T.C. E.N.Y. was supported by a Grass Foundation Fellowship.
We thank Dr. J. Byrne for the use of his osmometer.
Correspondence should be addressed to Ebenezer N. Yamoah at his present
address: Department of Physiology, Johns Hopkins University School of
Medicine, 725 North Wolfe Street, Baltimore, MD
21205-2185.
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