Brain Research, 481 (1989)399-402
Elsevier
399
BRE 23400
Enhancement of kainate-gated currents in retinal horizontal cells by
cyclic AMP-dependent protein kinase
Emily R. Liman*, Andrew G. Knapp and John E. Dowling
Department of Cellular and Developmental Biology, The Biological Laboratories,
Harvard University, Cambridge, MA 02138 (U.S.A.)
(Accepted 15 November 1988)
Key words: Retina; Horizontal cell; Kainate; CyclicAMP-dependent protein kinase
Dopamine, acting via cyclic adenosine 3':5'-monophosphate (cAMP), has been shown to enhance a kainate-gated ionic conductance in white perch retinal horizontal cells in vitro. To determine whether this effect involvesstimulation of a protein kinase, kainategated currents were observed in cultured horizontal cells that were dialyzed with the catalytic subunit of cAMP-dependent protein kinase. Intracellular application of catalytic subunit or cAMP, but not heat-inactivated catalytic subunit, caused significant enhancement of the kainate-evoked currents. These results suggest that kainate-gated channels in horizontal cells may be modified by a phosphorylation event.
Horizontal cells are second-order neurons that mediate lateral and feedback interactions in the outer
retina. In the teleost retina, cone-driven horizontal
cells receive two synaptic inputs: a (presumed) glutamatergic input from cone photoreceptors 14A8 and a
dopaminergic input from interplexiform cells 4&35. In
the white perch (Roccus americana), exogenously
applied dopamine has been found both to enhance
the sensitivity of cells to kainate 16 and to decrease the
degree of electrical coupling between neighboring
cells tT'19,2L29. These changes may contribute to the
regulation of horizontal cells' responsiveness in light
and darkness 2°,21,33,34.
The biochemical mechanisms mediating the actions of dopamine are not yet fully understood, but
there is good evidence for the involvement of cAMP.
Dopamine receptors are coupled to adenylate cyclase in the teleost retina 7,23,28,3°,31 and dopamine's
effects on both the kainate-gated 16 and the gap junctional 19'25'28 conductances are mimicked by analogs
and/or promotors of cAMP. Injection of cAMP-dependent protein kinase into white bass horizontal
cells causes them to uncouple electrically 17, implying
that gap junctional channels are modulated by phosphorylation. This report set out to determine whether the enhancement of kainate currents by dopamine
is likewise due to activation of cAMP-dependent protein kinase.
Retinal horizontal cells were isolated and cultured
according to previously published methods 6. Recordings were made from cells that had been in culture for
1-10 days. H2 cells, large luminosity-type conedriven cells with short thick processes were used. Recordings were made using the patch clamp method 12
in whole cell mode. Ringer's solution containing (in
mM): NaC1 145, KCI 2.5, N a H C O 3 20, glucose 10,
CaCI 2 2.5, MgSO 4 1 was equilibrated with 97%
02/3% CO2 and continuously circulated through the
culture dish. Electrodes were filled with (in mM) potassium gluconate 72, KF 48, E G T A 11, CaCI 2 1,
KC1 4, MgATP 0.01 and HEPES 10. Catalytic subunit of cAMP-dependent protein kinase (Sigma) was
mixed with the pipette solution at concentrations of
0.25 or 0.12 pM. The solutions containing catalytic
* Present address: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, U.S.A.
Correspondence: J.E. Dowling, The Biological Laboratories, Harvard University, Cambridge, MA 02138, U.S.A.
0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
400
subunit were used within 4 h of their preparation. Pipette solutions were stored at 4 °C whereas recordings were made at room temperature.
Kainate currents were evoked by pressure ejection
of Ringer's containing 5 0 / , M kainate from a small
bore pipette positioned aproximately 100 u m from
the cell. Cells were voltage clamped at - 6 0 mV and
ramp I - V curves 24 ( + 90 mV) were d e t e r m i n e d before and at the p e a k of the kainate current. The amplitude of the kainate current was obtained by subtracting the current at - 6 0 mV before kainate application from that at the peak of activation by kainate.
Kainate was first applied shortly after achieving
whole-cell voltage-clamp and at 1 rain intervals
thereafter. For the analysis, the maximum kainate
current attained was c o m p a r e d with the first kainate
current recorded for that cell, and the percentage increase over the first current was calculated. Thus, unless the first current was the largest, the control cells
as well as the experimental cells should show an increase in kainate currents. To evaluate the significance of the results, the experimental cells were comp a r e d (Student's t-test) to the controls rather than to
themselves. Cells used in the analysis were those
which survived for more than three minutes, for
which the pipette series resistance was low ( < 10
Mfa) and for which the leak currents did not increase
during the recording.
Application of kainate (50/xM) e v o k e d inward currents of 0 . 5 - 3 . 0 n A that reversed at approximately
+15 mV. With control solution in the pipette, the
amplitudes of the kainate currents were very uniform
over a ten minute period (see Fig. 1; m e a n maximal
% increase = 17% _+ 6.3, n = l l ) . With 11.25/~M catalytic subunit in the pipette, the responses of cells to
repetitive applications of kainate showed dramatic
enhancements of up to 200% (mean maximal increase = 124% _+ 31.5, n = 74 P < 0.005). There was
no change in the reversal potential of the kainategated current when catalytic subunit was present, nor
any change in the resting I - V curve (Fig. 2). A
smaller effect was seen with 0.12/~M catalytic subunit
(mean = 47.5% + 18.1, n = 6, P < 0.05).
The enhancement of kainate currents induced by
the catalytic subunit of c A M P - d e p e n d e n t protein kinase was not seen to reverse during any of the recordings as was expected since the pipette represents an
infinite supply of the kinase. However, in one case a
cell which survived the removal of the patch pipette
containing the catalytic subunit was subsequently recorded from with a pipette not containing kinase.
The kainate-gated current was observed to be close
to the control level. To check for nonspecific effects,
kinase solutions were boiled for 5 minutes and then
returned to 4 °C. There was no enhancement of kainate currents in cells perfused with these solutions
(mean = 12.3% + 9.1, n = 3, P > 0.10).
A m e m b r a n e p e r m e a n t analog of c A M P , 8-bromoc A M P , has been shown to enhance kainate currents
in horizontal cells. Therefore, it was expected that
horizontal cells dialyzed with c A M P would also show
enhanced kainate currents. We observed that kainate currents were enhanced by 63.7% + 21.5 (n =
10, P < 0.05) in cells dialyzed with 4 0 / t M c A M P . No
effort was made to block phosphodiesterases, which
may explain why the currents were not enhanced fur-
a
L./~
L/~
b
a~~"
4.0nA
2.0nA[
10 sec
Fig. 1. Whole-cell currents evoked by repeated applications of 50/~M kainate. The first response in each record was obtained < 1 min
after achieving the whole-cell recording configuration. The intracellular solutions were (a) control, (b) 0.25/~M catalytic subunit. The
rapid deflections in (b) indicate times when whole-cell I - V curves were determined (see Fig. 2).
401
I (nA)
÷1.0
V (mV)
Controls
0.5 rain
1 min
2 rain
- -3.0
Fig. 2. Whole-cell 1-V curves obtained by applying voltage ramps (+ 90 mV from a holding potential of-60 mV, 0.72 mV/ms) to the
cell in Fig. ld (0.25 #M catalytic subunit in the pipette). The three superimposed traces ('controls') were obtained between applications of kainate and represent the resting 1-V relation for this cell. The other traces were obtained at the peak of successive responses
to 50/~M kainate. The intersection of each of these traces with the control traces is the reversal potential of the kainate-gated current.
Times are relative to the start of whole-cell recording.
late the kainate-gated channel directly or to phosphorylate an effector of the channel. It will also be of
interest to determine in detail how phosphorylation
modulates the kainate-gated currents. In other preparations, cAMP-dependent processes have been
shown to affect a number of different channel properties including the number of functional channels 1'm'11'26'27,32, the probability of being open 1,2,
3,8,9,22, and the rate of desensitization ~3. Whatever
the mechanism(s) at work here, it has now been demonstrated that channels gated by excitatory amino
acids can be modified in at least two ways: allosterically, as in the enhancement of N M D A - g a t e d channels by glycine aS, and through a c A M P cascade, as in
the present case.
ther. Larger increases ( > 100%) in kainate currents
were observed in two cases in which pressure was
used to inject the intracellular solution. However, we
found this method to be unreliable. For instance, the
seal between pipette and cell was often broken by
such treatment. No enhancement of kainate currents
was observed in two cells in which c A M P but no ATP
was included in the intracellular solution. This experiment suggests that enhancement of kainate currents by c A M P involves a phosphorylation step that
requires ATP.
The results presented here are consistent with the
earlier report 16 that dopamine enhances kainate
gated conductances by acting through the stimulation
of adenylate cyclase. Further, the present report
demonstrates that cAMP-dependent protein kinase
can enhance kainate currents, suggesting that it is an
intermediary in the dopamine effect. It remains to be
seen whether the action of the kinase is to phosphory-
This work was supported by National Institutes of
Health Grants E Y 00824, E Y 05885, M H 14275 and
by the Charles A. King Trust, Boston, M A (A.G.K.).
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