Neural Science, Indiana University, Bloomington, Indiana 47405-7007
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ABSTRACT |
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Huang, Haojiang and Joseph Farley. PP1 Inhibitors Depolarize Hermissenda Photoreceptors and Reduce K+ Currents. J. Neurophysiol. 86: 1297-1311, 2001. Previous research indicates that activation of protein kinase C (PKC) plays a critical role in the induction and maintenance of memory-related changes in neural excitability of Type B photoreceptors in the eyes of nudibranch mollusk Hermissenda crassicornis (H.c.). The enhanced excitability of B cells is due in part to PKC-mediated reduction in somatic K+ currents. Here we examined the effects of protein phosphatase inhibitors on Type B photoreceptor excitability and K+ currents to determine the role(s) of protein phosphatases on memory formation in Hermissenda. Using electrophysiological and pharmacological methods, we found that the PP1 inhibitors calyculin A and inhibitor-2 depolarized Type B photoreceptors by 20-30 mV. A broad-spectrum kinase inhibitor, H7, blocked this effect. The depolarization induced by PP1 inhibition occluded that produced by an in vitro associative conditioning procedure. Calyculin and inhibitor-2 reduced the same B cell K+ currents (IA and Idelayed) that are reduced by in vitro and behavioral conditioning. H7 blocked the reductions. Cantharidic acid (PP2A inhibitor) and cyclosporin (PP2B inhibitor) had negligible effects on B cell resting membrane potential, K+ currents, and in vitro conditioning-produced cumulative depolarization of B cells. These results suggest that the functional activity of K+ channels in B cells is sustained by basal activity of PP1. Inhibiting PP1 appears to allow one or more constitutively active kinase(s) to reduce K+ channel activity and thus mimic the effects of conditioning. Our results suggest that PP1 may oppose and/or constrain the extent of learning-produced changes in B cell excitability.
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INTRODUCTION |
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Learning- and
memory-related modulation of synaptic plasticity and neuronal
excitability often involves posttranslational modifications of
receptors, ion channels, and synaptic vesicle proteins. Protein
phosphorylation and dephosphorylation is perhaps the most ubiquitous
and best-studied posttranslational mechanism by which protein function
can be altered (Cohen 1989; Krebs 1994
; Levitan 1999
; Nestler and Greengard
1999
). Indeed, elucidation of the cellular mechanisms
underlying learning and short- and intermediate-term forms of memory
has profited from the characterization of how activation of
second-messenger systems result in persistent modulation of membrane
ion channels, receptors, and other cellular processes by
phosphorylation (Bliss and Collingridge 1993
;
Dudai 1989
). Similarly, a prominent theme in studies of
long-term memory is cellular regulation of gene expression and protein
synthesis, which is often controlled by
phosphorylation/dephosphorylation of transcriptional activators and
repressors (Silva et al. 1998
). Although much early
research on the roles of phosphorylation and dephosphorylation in
learning and memory was focused on various protein kinases, interest in
the involvement of protein phosphatases has accelerated in recent years
(Asztalos et al. 1993
; Blitzer et al.
1998
; Endo et al. 1995
; Mulkey et al.
1994
).
A variety of lines of evidence indicate that persistent changes in
ocular Type B and A photoreceptor excitability contribute to
associative modifications of the sea snail Hermissenda
crassicornis's (H.c.) phototactic behavior, produced
by repeated pairings of light and rotation (Crow and Alkon
1978; Farley and Alkon 1980
, 1982
). These
persistent excitability changes have, in turn, been linked to
phosphorylation-mediated changes in K+ channel
activity, with the protein kinase C (PKC) (Crow and Forrester 1993
; Etcheberrigaray et al. 1992
; Farley
and Auerbach 1986
; Farley and Schuman 1991
),
protein tyrosine kinase (PTK) (I. Jin and J. Farley, unpublished data),
and MAP kinase (Crow et al. 1998
) families receiving the
greatest attention. Activators of PKC (Farley and Auerbach
1986
) or PTKs (Jin and Farley, unpublished results) mimic many
of the learning-produced changes in Type B cell excitability, expressed
as enhanced photoresponses, reductions in
IA and
IK-Ca, and decreases in resting
membrane conductance. Similarly, PKC activation by phorbol esters
precludes the occurrence of additional excitability changes in B cells
by in vitro conditioning (Jin and Farley, unpublished data; M. Smith,
I. Jin, R. McEwen, H. Huang, and J. Farley, unpublished
observations) or serotonin stimulation (Farley and Auerbach
1986
), suggesting that these three ways of increasing B cell
excitability converge biochemically. Conversely, prior enhancement of
the Type B cell photoresponse and input resistance by behavioral
conditioning (Smith et al., unpublished results) or serotonin
stimulation (Farley and Auerbach 1986
) attenuates the
increases produced by PKC activation (Smith et al., unpublished observations). Broad-spectrum kinase inhibitors such as H7
[1-(5-isoquinolinesulfonyl)-2-methylpiperazine], as well as those
more specific to PKC [such as sphingosine and staurosporine
(Farley and Schuman 1991
) and the peptide inhibitor PKC
(19-31) (Jin and Farley, unpublished data)], and PTKs [genistein, lavendustin A (Jin and Farley, unpublished results)] block in vitro
conditioning effects in B cells. Behavioral and in vitro conditioning
have both been reported to result in translocation and apparent
increases in PKC concentration/activity within the somatic membranes of
Type B cells (Impey et al. 1991
; McPhie et al.
1993
; Muzzio et al. 1997
).
In contrast to the evidence implicating phosphorylation events in
memory formation in Hermissenda, the involvement of
dephosphorylation has been relatively unexplored. One hint that
dephosphorylation might regulate the same K+
currents in Type B cells that are modulated by learning and kinase activation comes from a prior study of the ability of kinase inhibitors to prevent as well as reverse, conditioning-produced suppression of
K+ currents (Farley and Schuman
1991). This study reported that exposure of Type B cells (after
conditioning had already occurred) to the kinase inhibitors H7 or
sphingosine resulted in a relatively rapid (~30-60 min) reversal of
the K+ current suppression that had been produced
by behavioral conditioning but did not affect the
K+ currents from untrained B cells. These results
suggested an involvement of persistent kinase activity in
learning-produced K+ current suppression in Type
B cells, and also hinted at the presence of a highly active phosphatase
in B cells, whose activity was unmasked when kinase activity was suppressed.
Muzzio et al. (1999) have recently suggested that
phosphatase activation is responsible for massed trial learning
deficits in Hermissenda. When pairings of light and rotation
are administered at relatively short inter-trial intervals (ITIs),
phototactic suppression of Hermissenda and facilitation of B
cell excitability are reduced relative to more distributed training
regimens (Farley 1987a
; Farley and Alkon
1987
; Muzzio et al. 1999
). Increases in resting
intracellular Ca2+
concentration
[Ca2+]i
have
been suggested to mediate the massed-trial learning deficits, in part
through enhanced adaptation of photoreceptor light responses (Farley 1987b
; Farley and Alkon 1987
).
Muzzio et al. (1999)
found that basal
[Ca2+]i levels increased
when short ITIs separated successive light steps. This
[Ca2+]i accumulation
appeared to activate protein phosphatases because facilitation of Type
B photoreceptor excitability could be rescued in animals trained with
short ITIs if training occurred in the presence of okadaic acid (OA).
Thus Muzzio et al. (1999)
suggested that a PP2B-PP1/PP2A
activation scheme, similar to that proposed by Mulkey et al.
(1994)
to explain induction of hippocampal LTD, might be
responsible for massed-trial learning deficits in
Hermissenda.
To explore the potential contribution of dephosphorylation events to Type B cell membrane excitability, K+ channel activity, and learning in Hermissenda, Type B cells of untrained specimens were exposed to several widely used cell-permeant, as well as impermeant, inhibitors of serine/threonine protein phosphatases. We found that exposure of B cells to either calyculin A (an inhibitor of PP1 and PP2A) or inhibitor-2 (a specific peptide inhibitor of PP1) resulted in pronounced depolarization of B cells and suppression of the same K+ currents that are reduced by associative conditioning. These effects of calyculin A and inhibitor-2 were blocked by the kinase inhibitor H7, suggesting that inhibition of PP1 allowed a constitutively active protein kinase to dominate in phosphorylation/dephosphorylation cycles. Calyculin A and inhibitor-2 exposure also occluded in vitro conditioning produced changes in Type B photoreceptors, suggesting that PP1 normally serves to limit the extent of learning-produced changes in B cells. In contrast, cantharidic acid (PP2A inhibitor) and cyclosporin (PP2B inhibitor) had negligible effects on B cell resting membrane potential, input resistance, and K+ currents.
Portions of this research have previously been reported in abstract
form (Huang and Farley 1996, 1997
, 1999
).
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METHODS |
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Animals
Adult Hermissenda were provided by Sea Life Supply
(Sand City, CA). All animals were individually housed in perforated
50-ml tubes in artificial sea water (ASW) aquaria at 15°C on a 12-h light/dark cycle as previously described (Farley 1988).
Animals were fed with small pieces of Mytilus edulis every
other day.
Nervous-system preparation
The circumesophageal nervous system was dissected from an animal and placed on a glass microscope slide, within a ~400-µl well of standard ASW. Each preparation was incubated in 1 mg/ml of protease (Sigma type XXVII, catalog No. 4789) for ~10 min at room temperature (18°C) to facilitate cell impalement. After incubation, the nervous system was washed with a minimum of six volumes of 15°C ASW.
Intracellular recording and in vitro conditioning
Intracellular recording from an isolated nervous system was
effected using methods described previously (Farley and Alkon 1987). Glass microelectrodes (A and M Systems, catalog No.
6020) filled with 3 M KCl (30-40 M ohms) were used to impale cells. A
silver/silver chloride wire was used to connect the electrode solution
to the head stage, and a similar wire was used to ground the bath. All
recordings were made with an Axoclamp 2A (Axon Instruments) amplifier
in current-clamp mode, and appropriate head stages. Signals were
PCM-digitized at 44 MHz with the use of a Neurocorder (NeuroData
Instruments, No. 284) and stored on video tapes.
The input resistances of Type B photoreceptors were measured 1-2 min
after initial impalement, 2 min before the beginning of in vitro
conditioning, and 2-3 min after the end of in vitro conditioning.
Input resistances were measured from the voltage drops occurring across
the cell's membrane in response to injections of 0.5- to +0.2-nA
current steps (in 0.1-nA increments) through a balanced bridge circuit.
However, in many conditions, due to the presence of a drug or peptide
in the electrode, it was not possible to keep the bridge in balance
during current injection because of the high resistance or plugging of
the electrode. Resistance measurements were not obtained in these
cases. All recordings were obtained at room temperature (~18°C) in
standard ASW with the following composition (in mM): 430 Na+, 10 K+, 10 Ca2+, 50 Mg2+, 10 Tris HCl,
and 570 Cl
, pH = 7.6-7.8.
Prior to in vitro conditioning (Farley 1987b;
Farley and Alkon 1987
), a Type B photoreceptor and an
ipsilateral statocyst caudal hair cell were impaled, and the
preparation was dark-adapted for 10 min. During in vitro conditioning,
the nervous system was exposed to five 30-s simultaneous presentations
of whole-field illumination (at an intensity of ~300
µW/cm2) and depolarizing current stimulation of
the hair cell (0.1-0.5 nA) every 2 min. The membrane potential of the
B cell was continuously monitored for
5 min after conditioning. The
2-min postconditioning value is reported here. Peak- and steady-state
light responses were also measured (to the nearest mV) during the first
three light steps given following the ten min dark-adaptation period.
For those preparations and drug conditions in which the resting membrane potential of the Type B cell was stable (<2 mV change during entire dark-adaptation period, <0.50 mV change during the last 5 min of dark adaptation), cumulative depolarization of the B cell was measured by comparing the membrane potential 2 min after the last conditioning trial with the resting membrane potential value measured just before the first trial. However, cells exposed to calyculin A or inhibitor-2 often continued to depolarize throughout dark-adaptation, and a stable preconditioning membrane potential value could not be ascertained. In these cases, the membrane potential value was extrapolated to 2 min after the last conditioning trial, assuming that the B cell would have continued to depolarize at the same rate as had occurred during the 10 min prior to conditioning. Cumulative depolarization for these cells was measured by comparing the actual membrane potential 2 min after the last trial to the extrapolated membrane potential at that point (e.g., see Fig. 4B).
The reliability of these extrapolated membrane potential values was assessed as follows. In seven separate control experiments in which preparations were exposed to calyculin A but were not exposed to in vitro conditioning, we estimated the rate of membrane depolarization during the 10-min dark-adaptation period. We then extrapolated the membrane potential value during the next 12 min, the time when five pairings of light and hair cell stimulation would have been administered had these preparations been exposed to in vitro conditioning. We then compared the actual membrane potential with the extrapolated value (extrapolated minus actual) and found that these differed from each other by an average of 0.29 ± 0.46 (SE) mV. Thus the extrapolated values provided reasonably accurate estimates of the actual membrane potential 12 min later and validated the use of extrapolated values for the purposes of assessing the extent of cumulative depolarization.
Drugs
Calyculin A, inhibitor-2, and cantharidic acid were obtained from Calbiochem. H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], H8 {N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, HCl} and cyclosporin A were obtained from Sigma.
Calyculin A was dissolved in dimethylsulfoxide (DMSO) at an initial concentration of 0.1 mM. The drug was then diluted in distilled water to a 2 µM stock concentration. When either bath-application or intracellular leakage was used to deliver the drug, calyculin A was added to the ASW bath or microelectrode 3 M KCl solution to a final concentration of 20 nM (final DMSO concentration was 0.02%). When introduced directly into Type B cells, cantharidic acid was dissolved at a concentration of 1 µM in 3 M KCl solution (0.01% DMSO). Similarly, cyclosporin A was prepared at a concentration of 100 nM in 3 M KCL (0.01% DMSO). Inhibitor-2 was dissolved in 3 M KCl at a concentration of 20 nM, and the peptide was allowed to leak into Type B cells. H7 was dissolved in ASW at a concentration of either 60 or 120 µM. H8 was dissolved in ASW at a concentration of 150 µM. Stock solutions of phosphatase and kinase inhibitors were made fresh, every day or two, and care was taken to prevent photoinactivation of light-sensitive compounds.
In control experiments, preparations were bathed in normal ASW. The
concentrations used for the protein phosphatase inhibitors [calyculin A (PP1 and PP2A inhibitor), inhibitor 2 (PP1
inhibitor), cantharidic acid (PP2A inhibitor), and cyclosporin A (PP2B
inhibitor)], were 10-40 times higher than the
IC50 values reported for inhibition of the
respective phosphatases in purified enzyme assays and were very similar
to those used by others in electrophysiological experiments (Furukawa et al. 1996; Holm et al. 1997
;
White et al. 1993
). In most experiments, a drug was
allowed to leak into a Type B cell through relatively low resistance
electrodes (10-15 M
) filled with inhibitor and 3 M KCl. In several
sets of experiments, calyculin A was applied in the bath (20 nM).
Voltage clamp
All experiments were performed on Type B cell somata that had
been isolated by axotomy from all synaptic interactions, as well as
from any impulse-generating membrane, as previously described (Alkon and Fuortes 1972; Farley and Auerbach
1986
; Farley and Han 1997
). The axons of the
photoreceptors leave the base of the eye, where they enter the optic
nerve, and pass ensheathed through the optic ganglion (Eakin et
al. 1967
; Stensaas et al. 1969
). The
photoreceptor axons within the optic nerve join the optic tract and
terminate in a spray of fine endings within the cerebropleural neuropil, ~100 µm from their somata (Auerbach et al.
1989
; Eakin et al. 1967
; Senft et al.
1982
). The optic nerve was razor-lesioned at the point where it
exited the base of the eye, proximal to its entry into the
cerebropleural neuropil. Such a lesion leaves a photoreceptor cell body
(~35-50 µm in diameter) that contains the rhabdomeric
phototransduction apparatus and responds to light with normal generator
potentials but is without action potentials or detectable synaptic
interactions (Alkon and Grossman 1978
; Farley and Auerbach 1986
; Farley and Han
1997
).
Standard two-electrode voltage-clamp methods were used (Farley
and Auerbach 1986; Farley and Han 1997
). The
electrodes used to monitor membrane potential had resistances of 30-40
M
when filled with 3 M KCl. A lower resistance electrode (10-15
M
) was used for current passage and drug leakage. Data acquisition
and analysis were performed using Axon Instrument's pClamp 5.5 and 6.0 program suites. A Type B cell was accepted for further study if the
resting membrane potential was more negative than
40 mV and the
steady-state light response to a 300 µW/cm2
light step was more than 12 mV. Unless otherwise indicated, holding potential was
60 mV. Four-hundred-millisecond depolarizing command steps were used to elicit voltage-dependent K+
currents. The stimulation frequency used (every 7 s) resulted in
no appreciable cumulative inactivation. Activation time constants (Tauon) for individual
IA current traces were fit
assuming a power function
A(1
e
(t
K)/Tauon)n + B
, where n = 3, A = final current amplitude, B = vertical offset.
Inactivation time constants (Tauoff) were fit
with single-exponential functions
A*e
(t
K)/Tauoff + B
. The goodness-of-fit values
(R2) for all reported time constants
were >0.95.
Statistical analysis
Differences in membrane potential, input resistance, cumulative depolarization, light responses, and ionic current amplitudes produced by different treatment conditions were assessed using appropriate analyses of variance (ANOVA). When a significant F value was obtained, post hoc multiple-comparisons (Tukey's HSD test) were used when comparing three or more treatment means to each other and/or a control condition. In the absence of ANOVA, a simple Student's t-test was used to compare the effects of two treatment conditions. Two-tailed significance tests were used and are reported as significant if P < 0.05, unless otherwise indicated.
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RESULTS |
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PP1 inhibitors depolarize Type B cells and increase resting input resistance
To determine a role for constitutive protein phosphatase activity
in regulating the excitability of Type B photoreceptors, 20 nM
calyculin A, a PP1 and PP2A inhibitor, was applied to the bath, and
changes in resting membrane potential (Fig.
1) and input resistance of synaptically
intact B cells were measured. Because of the variable duration of
recording across experiments following drug application, which was
never <10 min, the effects on resting membrane potential are given as
rates of depolarization. Over a 10- to 30-min recording period,
calyculin A depolarized B cells at a rate of 0.8 ± 0.12 (SE)
mV/min (Fig. 1, A and B) and increased resting
input resistance by 24 ± 8% (n = 7). The
depolarization produced by calyculin was quite dramatic with cells
changing by 20-30 mV during 20-30 min exposure to calyculin.
Consistent with the effects of other agents that produce large
depolarizations of Type B cells (e.g., high extracellular
K+) (Alkon et al. 1984;
Farley 1988
), cells that were exposed to calyculin for
20-30 min and allowed to fully depolarize until their membrane
potential stabilized (range of about
25 to
15 mV), showed markedly
abnormal (slow and small) light responses and generally lost their
ability to spike.
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To determine how much of the calyculin-produced depolarization was attributable to inhibition of phosphatase activity within the B cell itself, as opposed to processes presynaptic to B cells, the effects of bath-application of calyculin were compared for synaptically intact (calyculin bath condition, Fig. 1B) versus isolated B cells (isolated calyculin condition, Fig. 1B). The rate of depolarization was 0.72 ± 0.15 mV/min for synaptically-isolated cells (Fig. 1B), which was not significantly different from that of intact cells [t(25) = 0.48].
When calyculin A was allowed to leak into synaptically intact B cells, it depolarized the B cells at a rate of 0.99 ± 0.24 mV/min (calyculin leak condition; Fig. 1B). This rate was slightly, though not significantly [t(29) = 0.78], greater than that of bath application. These results indicate that bath-applied calyculin depolarized B cells primarily through mechanisms endogenous to the B cells, such as inhibition of PP1 and/or PP2A.
To further differentiate between PP1 and PP2A as the target(s) for calyculin, inhibitor-2, a selective peptide inhibitor of PP1, was allowed to leak into Type B photoreceptors. Inhibitor-2 depolarized B cells at a rate of 0.88 ± 0.13 mV/min (Fig. 1B), which was not significantly different from calyculin bath-application [t(30) = 0.23] or calyculin leakage [t(17) = 0.47]. Other phosphatase inhibitors, such as cantharidic acid (a more specific PP2A inhibitor) and cyclosporin A (a specific PP2B inhibitor) produced negligible depolarization (<1.5 mV over 10 min; Fig. 1B).
The effects of phosphatase inhibitors on the light responses of Type B photoreceptors were determined by measuring both the peak and steady-state components of the light response for different cells in the presence versus absence of inhibitors following a standard 10 min dark-adaptation period. The results for three consecutive light steps, delivered at a 2.0-min inter-stimulus interval (ISI), were averaged. In general, Type B cells exposed to the phosphatase inhibitors tested here showed smaller peak and steady-state depolarizing generator potentials than control condition cells (Fig. 2), but only some of these differences were significant.
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An ANOVA of the peak light-induced response data for all conditions revealed a significant effect of drug [F(5,49) = 8.91, P < 0.01], with intracellular leakage of calyculin [Tukey's HSD post hoc test: t(15) = 4.23, P < 0.01], and inhibitor-2 [t(16) = 5.87, P < 0.01] producing significant reductions of peak light response when compared with controls. The peak light responses of cells exposed to bath-applied calyculin, leakage of cyclosporin, or leakage of cantharidic acid were not significantly different from controls [t(24) = 2.24, t(9) = 2.07 and t(9) = 2.15, respectively]. Similarly, an ANOVA of the light-induced steady-state generator potential data for all conditions revealed a significant effect of drug [F(5,49) = 11.28, P < 0.01]. Bath-applied calyculin [t(24) = 4.25, P < 0.01], and intracellular leakage of calyculin [t(15) = 3.89, P < 0.01], inhibitor-2 [t(16) = 7.23, P < 0.01], and cantharidic acid [t(9) = 3.94, P < 0.01] produced significant reductions of the steady-state light response when compared with controls. The steady-state light response of cells exposed to cyclosporin was not significantly different from controls [t(9) = 1.89].
These results indicate a partial dissociation between the effects of phosphatase inhibitors on resting membrane excitability and light responses of B cells. PP1 inhibitors (calyculin and inhibitor-2) affected both. Cantharidic acid (PP2A inhibitor) reduced the steady-state light response without causing appreciable depolarization. Cyclosporin (PP2B inhibitor) failed to affect the resting membrane potential and produced a slight (though nonsignificant) reduction in the light response. The reduction of the light response by cantharidic acid is noteworthy in two respects. First, it suggests that in addition to PP1, PP2A might also regulate phototransduction processes in Type B cells. Second, the reduction indicates that the failure of cantharidic acid to depolarize Type B cells cannot be attributed to a failure of drug delivery. Despite the fact that cantharidic acid reduced both components of the light response by approximately the same amount as bath-applied calyculin and was thus clearly delivered to cells, the effects of calyculin on resting membrane potential were about seven to eight times greater than those of cantharidic acid. A similar, though weaker case, can be made for PP2B. Although cyclosporin failed to significantly reduce the light response of B cells when evaluated using relatively conservative post hoc Tukey tests, Student's t-tests indicated significantly smaller peak [t(9) = 2.49, P < 0.05], but not steady-state [t(9) = 1.65] light responses, when compared with controls. But cyclosporin, like cantharidic acid, had no effect on B cell resting membrane potential.
Collectively, our results suggest that PP1 is a primary serine/threonine protein phosphatase in B cells that is affected by calyculin A and inhibitor-2. Further, constitutive PP1 activity is involved in regulating the excitability of B cells.
H7 blocks the effects of calyculin A and inhibitor-2
Preexposure of B cells to the broad-spectrum S/T kinase inhibitor H7 blocked the effects of calyculin A and inhibitor-2 in a dose-dependent fashion (Fig. 3). In the presence of 60 µM bath-applied H7, the average rate of depolarization caused by leakage of calyculin A (~1.0 mV/min) was reduced by 43% (0.56 ± 0.15 mV/min). When a higher concentration of H7 was used (120 µM), the effects of bath-applied calyculin and intracellular leakage of inhibitor-2 were reduced by 94% (0.05 ± 0.05 mV/min) and 89% (0.1 ± 0.1 mV/min), respectively. In contrast to H7, a high concentration of H8 (a potent and specific inhibitor of cyclic nucleotide-dependent protein kinases) produced only a partial block (18%) of calyculin's effect (n = 4).
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These data imply that the effects of calyculin A and inhibitor-2 were due to a disruption of the balance between phosphorylation and dephosphorylation rather than through direct effects on membrane excitability (e.g., block of resting K+ channels, activation of channels with a reversal potential more positive than rest, etc.). When PP1 was inhibited by either calyculin A or inhibitor-2, serine/threonine kinase(s) apparently dominated the phosphorylation/dephosphorylation cycles and led to increased phosphorylation. The depolarization and increased input resistance of B cells are consistent with phosphatase inhibition (increased phosphorylation) having produced a closure of somatic K+ channels.
PP1 inhibitors occlude conditioning produced cumulative depolarization of B cells
Isolated nervous systems from untrained Hermissenda
were conditioned in vitro in the presence or absence of protein
phosphatase inhibitors (Fig. 4). Five
pairings of light and statocyst hair-cell stimulation in standard ASW
resulted in a cumulative depolarization of 6.86 ± 0.4 mV
(n = 7; Fig. 4, A and D)
consistent with that observed in previous studies (Farley
1987b; Farley and Alkon 1987
; Farley and
Schuman 1991
; Grover and Farley 1987
). When
conditioned in the presence of either bath-applied calyculin A
(n = 5; Fig. 4, B and D),
intracellular leakage of calyculin A (n = 8; Fig. 4,
C and D), or intracellular leakage of inhibitor-2
(n = 6; Fig. 4D), Type B cells showed
substantially and significantly reduced cumulative depolarization. In
fact, in vitro conditioning following intracellular leakage of
calyculin or inhibitor-2 produced nominal net hyperpolarizations:
1.13 ± 1.11 and
1.08 ± 1.38 mV, respectively (Fig.
4D). Whether these small hyperpolarizations are genuine or
not is unclear. The reference membrane potentials for the majority of
these experiments were extrapolated values, and an error in extrapolation could result in an artificial apparent hyperpolarization.
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However, in three of these experiments (2 involving
calyculin-leakage and 1 involving bath-applied calyculin), the
depolarization produced by calyculin was either negligible, or had
ceased, by the end of the dark-adaptation period when in vitro
conditioning was initiated. Thus the stable resting membrane potentials
of these B cells during the 1-min period prior to in vitro conditioning were used as the reference potentials (e.g., Fig. 4C). For
these three preparations, in vitro conditioning produced only 0.33 ± 0.41 mV of depolarization. This was significantly less than that of
control preparations [t(8) = 11.28, P < 0.0001] but not different [t(11) = 0.30] from the
combined average hyperpolarization of 0.30 ± 1.15 mV
(n = 10) observed for the majority of the
calyculin-leakage and -bath-treated cells whose membrane potentials had
not stabilized by the start of in vitro conditioning. Thus attenuation
of cumulative depolarization by calyculin was not specific to cells
whose reference potentials were extrapolated values. And it seems safe
to conclude that calyculin- and inhibitor-2-treated cells were not
depolarized as much by in vitro conditioning as controls.
In contrast to the effects of PP1 inhibitors, cantharidic acid (n = 4) and cyclosporin A (n = 4) failed to affect cumulative depolarization (Fig. 4D). In the presence of these PP inhibitors, in vitro conditioning produced depolarizations similar to that of controls (cantharidic acid: 7.1 ± 1.1 mV; cyclosporin A: 7.9 ± 1.6 mV, respectively).
An ANOVA of the cumulative depolarization data for all treatment conditions revealed a significant drug effect [F(5,28) = 12.6, P < 0.01], with bath-applied calyculin [t(10) = 3.29, P < 0.05], intracellular leakage of calyculin [t(13) = 5.34, P < 0.01], and intracellular leakage of inhibitor-2 [t(11) = 4.93, P < 0.01] producing significant reductions of cumulative depolarization as compared with controls. In contrast, the depolarizations produced by in vitro conditioning for cells exposed to cyclosporin or cantharidic acid were not significantly different from controls [t(9) = 0.15 and t(9) = 0.56, respectively].
Thus prior depolarization of B cells by the PP1 inhibitors calyculin
and inhibitor-2 occluded the cumulative depolarization resulting from
in vitro conditioning. This suggests that PP1 phosphatase inhibition
and behavioral conditioning share common biochemical mechanisms in
their regulation of B cell excitability. Because conditioning-produced
cumulative depolarization of B cells results from reduced
K+ channel activity (Farley 1987b;
Farley and Alkon 1987
; Farley and Schuman
1991
), the present findings suggest the hypothesis that
calyculin and inhibitor-2, through inhibition of PP1, lead to
phosphorylation and reduced activity of K+
channels in B cells. This in turn leads to depolarization and occlusion
of in vitro conditioning-produced depolarization of B cells.
PP1 inhibitors reduce B cell K+ currents
Two macroscopic K+ currents,
IA (a rapidly inactivating,
voltage-dependent "A type" current) and
Idelayed (a slow, noninactivating current) were recorded as in previous studies (Alkon et al.
1984; Farley 1987a
, 1988
; Farley and
Auerbach 1986
) (Fig.
5A). A major component of
Idelayed is a slow
Ca2+-activated K+ current
(IK-Ca) (Alkon et al.
1984
; Farley 1988
; Farley and Auerbach 1986
; ). In the absence of PP inhibitors,
IA and
Idelayed showed only slight rundowns
during extended recording periods, declining by ~8% over 30 min
(Fig. 5, B and C). Thirty minutes following addition of calyculin A to the bath,
IA and
Idelayed were reduced by 45 and 67%,
respectively (n = 7; Fig. 5, A-C).
Similarly, when inhibitor-2 was allowed to leak into the B cell,
reductions of 49 and 63% were recorded for
IA and
Idelayed, respectively
(n = 8; Fig. 5, A-C). In contrast, PP2A and
PP2B inhibitors had much smaller effects on these
K+ currents (Fig. 5, B and
C). Thirty minutes following the establishment of
voltage-clamp and initiation of drug-leakage, cantharidic acid had
reduced IA and
Idelayed by 25 and 23%, respectively
(n = 5). Similarly, cyclosporin A reduced
IA and
Idelayed by 26 and 13%, respectively
(n = 4).
|
An ANOVA of the peak K+ current amplitudes at 0 mV, 30 min following initiation of drug exposure, revealed a significant effect of drug [F(4,11) = 4.49, P < 0.05], with calyculin [Tukey's HSD post hoc tests: t(7) = 3.61], and inhibitor-2 [t(5) = 3.5] producing significant reductions of peak K+ currents when compared with controls. The peak currents of cells exposed to cantharidic acid, or cyclosporin, were not significantly different from controls [t(4) = 1.23 and t(4) = 1.34, respectively]. A similar ANOVA and post hoc comparison of the delayed K+ current data revealed a significant drug effect [F(4,13) = 6.509, P < 0.01], with calyculin [t(7) = 3.86, P < 0.05] and inhibitor-2 [t(7) = 3.6, P < 0.05] producing significant reductions of delayed K+ currents when compared with controls. The delayed K+ current amplitudes from cells exposed to cantharidic acid or cyclosporin were not significantly different from controls [t(4) = 0.51 and t(4) = 0.06, respectively].
The reductions of K+ currents produced by PP1 inhibitors were blocked by 120 µM bath-applied H7 (Fig. 6).
|
In addition to reducing the amplitudes of
IA (Fig.
7, A and C) and
Idelayed (Fig. 7, B and
D) at potentials equal to or more positive than 25 mV, PP1
inhibitors also altered the voltage dependence of
Idelayed but not of
IA. Following exposure to inhibitor-2, the I-V curve for IA showed
a ~7- to 8-mV shift toward more depolarized membrane potentials (Fig.
7A), without appreciable change in
slope.1 In
contrast, the slope of the I-V curve for
Idelayed (Fig. 7B) was
clearly reduced by inhibitor-2. In the absence of inhibitor-2, IA and
Idelayed exhibited e-fold
increases per 14.36 ± 1.32 and 15.81 ± 0.93 mV,
respectively, when measured over the range of steepest
voltage-dependency. In the presence of inhibitor-2,
IA and
Idelayed showed
e-fold increases per 15.39 ± 0.84 and 18.93 ± 1.23 mV.
|
Similar to inhibitor-2, the voltage dependence of IA was not changed by calyculin A, with the I-V curve being shifted positive by ~10 mV (Fig. 8A). However, calyculin A reduced the slope of the I-V curve for Idelayed (Fig. 8B), with e-fold changes in current-amplitude requiring 19.94 ± 0.94 mV of depolarization in the presence of calyculin versus 16.92 ± 1.17 mV in its absence.
|
The activation (Tauon) and inactivation (Tauoff) kinetics of IA (Fig. 9A) were only weakly voltage dependent, showing only slight changes with membrane potential. Inhibitor-2 produced a small, nonsignificant increase (~15%) in Tauon (Fig. 9B), and a modest (~20%) but again nonsignificant decrease in Tauoff (Fig. 9C). On average, calyculin A also produced a small, nonsignificant increase (~12%) in Tauon (Fig. 9D) but a slight nonsignificant increase in Tauoff (Fig. 9E). In summary, inhibitor-2 and calyculin both produced a small but consistent slowing of activation of IA (increased Tauon), that may have contributed (slightly) to the reduction in IA amplitudes. In contrast, the effects of the PP1 inhibitors on Tauoff were inconsistent with each other and nonsignificant. Although it is possible that the modest and nonsignificant reductions in Tauoff produced by inhibitor-2 (i.e., faster inactivation of IA) may have contributed slightly to the reduction in peak amplitudes, it seems unlikely that this mechanism was the major one accounting for reduction in peak amplitudes. The slight slowing of inactivation rate produced by calyculin obviously cannot be the mechanism that produced reductions in peak amplitudes.
|
Functional equivalence of PP1-inhibition, PKC-activation, and conditioning
The effects of PP1 inhibitors on Type B cell
K+ currents were qualitatively very similar to
those of phorbol esters (PKC-activators) (Farley and Auerbach
1986; Smith et al., unpublished data), serotonin-stimulation (Farley and Auerbach 1986
; Farley and Wu
1989
) [which participates in the learning-induced plasticity
of B cells and occludes the effects of phorbol esters (Auerbach
et al. 1989
; Crow and Forrester 1991
;
Grover et al. 1989
)], in vitro conditioning of
isolated nervous systems (Farley and Schuman 1991
), and
multi-trial behavioral training of intact animals (Alkon et al.
1985
; Farley 1988
). However, there were
quantitative differences among these treatments in their effects on
K+ current amplitudes and voltage dependency. For
example, phorbol ester (Farley and Auerbach 1986
) and
serotonin-application (Farley and Wu 1989
) both reduce
IA peak amplitudes by ~30% over
most of the activation range but have little effect (~5-10%
reduction) on A-current voltage dependency. Similarly, multi-trial
behavioral conditioning of intact animals reduces peak
IA currents by ~30% (Alkon
et al. 1982
; Farley 1988
; Farley and
Schuman 1991
), produces a depolarizing shift of ~10 mV in the
I-V curve for IA but only slightly reduces (~5-10%) the voltage dependency of the A-type conductance (slope of I-V curve).
In contrast, injections of exogenous PKC into B cells reduced A
currents by 30-60%, depending on the activation potential, and
produced a corresponding flattening of the I-V curve
(Farley and Auerbach 1986). Similarly, in vitro
conditioning reduced IA by 30-60%,
depending on activation potential, and also produces a marked reduction
in voltage dependency (Farley and Schuman 1991
).
Thus manipulations that produce large IA reductions (in vitro conditioning, PKC injections) also reduce the voltage dependency of the conductance. Manipulations that produce less dramatic reductions in IA (serotonin stimulation, phorbol esters, behavioral conditioning, and PP1 inhibition) have correspondingly smaller effects on voltage-dependency. All manipulations that reduce IA appear to produce slight decreases in activation kinetics, but these do not appear to be correlated with the magnitude of IA suppression.
Although multi-trial behavioral conditioning reduces the peak
amplitudes of composite Idelayed
(Alkon et al. 1985) and pharmacologically isolated
IK-Ca (Farley 1988
;
Farley and Schuman 1991
) by ~50% over the activation
range, the effects on voltage dependency are generally much more
pronounced than for the A conductance. Typically, the slopes of the
I-V curves are reduced by >45%. Phorbol esters/PKC injections (Farley and Auerbach 1986
), and in vitro
conditioning (Farley and Schuman 1991
) also reduce the
voltage dependency by >45%.
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DISCUSSION |
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PP1 inhibitors affect B cell excitability
PP1 inhibitors, calyculin A and inhibitor-2, produced large (20-30 mV in 30 min) depolarizations of Type B photoreceptors, accompanied by an increase in resting input resistance. Approximately equivalent depolarizations were observed regardless of whether the inhibitors were applied in the bath or intracellularly through the recording electrode, and whether the cells were synaptically intact or isolated. Both results imply that the principal drug targets responsible for depolarization were endogenous to Type B cells. The depolarization resulting from PP1 inhibitors mimicked and occluded that produced by in vitro conditioning, suggesting that conditioning and PP1 inhibition affected a common signal transduction cascade. In contrast, cantharidic acid and cyclosporin, potent and specific inhibitors of PP2A and PP2B, had negligible effects on B cell resting membrane potential and failed to occlude in vitro conditioning.
Voltage-clamp analysis revealed that PP1 inhibitors suppressed
IA and
Idelayed of B cells.
IA's voltage dependency was
unaffected by PP1 inhibitors, with the I-V curve showing a
~7- to 10-mV parallel displacement toward depolarized potentials.
Activation kinetics were only slightly slowed (by ~12-15%) but may
have contributed somewhat to the reductions in peak current amplitudes.
In the case of Idelayed, the voltage
dependence of the macroscopic current was clearly reduced. Macroscopic
Idelayed is composed of residual IA,
IK,V, and
IK-Ca (Alkon et al.
1984; Farley 1988
; Farley and Auerbach
1986
; Farley and Schuman 1991
; Farley and
Wu 1989
), and their relative contributions to
Idelayed change as a function of
membrane potential. At potentials between
25 to +10 mV (inclusive), IK-Ca is the major contributor
accounting for
60% of macroscopic Idelayed (Alkon et al.
1984
; Farley 1988
). Thus it is likely that reductions of IK-Ca account for much
of the reduction in Idelayed. Additional analysis of the effects of PP1 inhibitors on
Idelayed will require isolation of the
different current components before clear conclusions can be drawn as
to mechanisms responsible for the apparent reduction in voltage
sensitivity. However, to a first approximation, the effects of PP1
inhibitors were very similar to those produced by behavioral
conditioning, and further reinforce the conclusion that PP1-inhibition
and associative training involve common biochemical mechanisms.
The broad-spectrum S/T-kinase inhibitor H7 blocked the membrane depolarization and K+ current suppression produced by PP1-inhibitors, implying that the inhibitors' effects probably resulted from perturbation of phosphorylation-dephosphorylation cycles rather than from some other process unrelated to phosphatase inhibition (e.g., a direct block of K+ channels).
Collectively, the present and previous results suggest the scheme for
kinase/phosphatase involvement in learning-produced changes in B cells
depicted in Fig. 10. Prior to
conditioning, the functional activity of "A type" and
Ca2+-activated K+ channels
in somatic membranes of B cells is sustained by constitutive PP1
activity, which evidently dominates any suppressive effects of basal
constitutive phosphorylation. Calyculin-A and inhibitor-2 treatments,
presumably through inhibition of PP1, allow either constitutively-active kinases (and/or induce activation of kinases) to
reduce K+ channel activities (Fig. 10). Because
the activities of these same K+ channels are
reduced by in vitro (Farley 1987b; Farley and
Schuman 1991
) and multi-trial behavioral conditioning of intact
animals (Alkon et al. 1982
, 1985
; Farley
1988
), through PKC- (Farley and Auerbach 1986
;
Farley and Schuman 1991
) and PTK-mediated (Jin and Farley 2001) phosphorylation events, the result is that PP1 inhibitors occlude the effects of conditioning.
|
Dephosphorylation and K+ channels
Protein phosphatases have been shown to regulate a wide variety of
ion channels (Herzig and Neumann 2000), especially
K+ channels. Because of the functional and
structural diversity of K+ channels, and the
different types of cell in which they have been studied, a common
scheme for phosphatase-linked regulation of K+
channels has not yet emerged. Nevertheless some of our results are very
similar to those of others.
In voltage-clamped Aplysia sensory neurons, intracellular
injections of phosphatase inhibitors, okadaic acid and microcystin-LR, altered baseline currents and occluded 5-HT and cAMP-induced reductions in K+ currents (Ichinose and Byrne
1991; Ichinose et al. 1990
). These studies
implicated PP1 and/or PP2A in the reversal of cAMP-induced phosphorylations. Conversely, injections of purified mammalian PP1 or
PP2A catalytic subunits mimicked the effects of FMRFamide and induced
outward K+ currents. Additional studies have
since narrowed the focus to PP1 (Endo et al. 1995
).
Dephosphorylation of Ca2+-activated
K+ channels (or associated proteins) has been
reported to increase their activity in a variety of nonneuronal cell
types (White et al. 1991, 1993
; Zhou et al. 1996
). Several examples of this type of regulation have
implicated a cGMP-PKG-PP2A signaling pathway. A similar mechanism may
also operate in neurons (Furukawa et al. 1996
;
Holm et al. 1997
). Similarly, Pedarzani et al.
(1998)
reported that the Ca2+-activated
K+ channels that underlie the slow
afterhyperpolarization (sIAHP) in rat
CA1 pyramidal neurons are slowly suppressed by inhibitors of PP1/PP2A.
A variety of large conductance Ca2+-activated
K+ channel (BKCa) subtypes,
isolated from mammalian brain synaptosomes and incorporated into
artificial bilayer membranes (Farley and Rudy 1988),
have been found to be differentially modulated by S/T kinases and
phosphatases. Type 1 channels are stimulated by PKA (Farley and
Rudy 1988
; Reinhart et al. 1991
) and inhibited
by PP2A (Reinhart et al. 1991
). Type 2 channels show the
reverse pattern of effects (Reinhart et al. 1991
).
Collectively, these results raise the possibility that stimulation of Ca2+-activated K+ channels by dephosphorylation through PP2A/PP1 may be a common mechanism to reduce cellular excitability and promote calcium homeostasis, thereby contributing to anti-apoptotic and neuroprotective functions.
In summary, both voltage- and Ca2+-activated
K+ channels are regulated in a variety of diverse
ways, including phosphorylation by PKG, PKC, and PKA and
dephosphorylation by PP2A and PP1. These enzymes appear to be tightly
associated with channel complexes and are often activated under basal
stimulation conditions (Reinhart and Levitan 1995). Thus
our findings that PP1 inhibitors shut K+ channels
in B cells, presumably by allowing a constitutively active kinase to
dominate, has several precedents in the literature.
Physiological role(s) of PP1 in B cells?
Our results suggest that conditioning and PP1-inhibition affect a
common signal transduction cascade in B cells. Our results do not,
however, define the precise physiological role for PP1. One interesting
possibility is that in addition to activation of PKC, concomitant
inhibition of PP1 may be necessary for the induction of (excitatory)
conditioning produced-changes in B cell excitability, similar to the
"gating" function proposed for PP1 inhibition in the transition
from the intermediate- to late-phase stages of LTP in the hippocampus
(Blitzer et al. 1998). PP1 may oppose and/or constrain
the extent of learning-produced changes in B cell excitability and thus
function as a checkpoint for memory formation.
One problem with this hypothesis as it applies to
Hermissenda concerns the phosphatase inhibitor-produced
large depolarizations of Type B cells, which were accompanied by the
loss of light responses and spiking. Prolonged 20- to 30-mV
depolarizations of B cells are likely to be lethal. It is quite likely
that [Ca2+]i levels in B
cells increased during these depolarizations, since the final
membrane potential reached (range of 25 to
15 mV) overlaps with
that for gating of voltage-dependent Ca2+
channels in B cells (Alkon et al. 1984
; Farley
1988
). Diminished light responses in B cells can be produced by
intracellular injections of Ca2+ (Alkon
1979
; Sakakibara et al. 1998
),
consistent with the widely accepted view of Ca2+
as a mediator of light adaptation in invertebrate photoreceptors (Minke and Selinger 1996
; Nagy 1991
).
Although B cells undergo a cumulative depolarization during
conditioning, the depolarization reaches a plateau of ~8-10 mV after
10 light-rotation pairings (Farley 1987a; Farley
and Alkon 1987
; Grover and Farley 1987
), far
less than the 20-30 mV produced by calyculin and inhibitor-2. And in
vitro-conditioned B cells retain their light response and spiking ability.
Thus a problem for the preceding PP1-inhibition hypothesis is that if such a process did occur for prolonged time periods, it would be expected to trigger apoptosis if not necrosis. Therefore it may be that PP1 is further activated during conditioning to constrain and limit the depolarization that results from pairing-produced PKC- and PTK-mediated reductions in K+ channel activity. This would be consistent with the general theme that has emerged from studies of PP1's effects on Ca2+-activated K+ channels reviewed earlier: PP1-activation of K+ channels results in hyperpolarization, reduced excitability, and protection against cell death.
It is also possible that PP1 activity might be further stimulated by
other training conditions. Because PP1 activity is associated with
increased K+ currents in B cells, it is
intriguing to consider the possibility that PP1 might be activated by
conditioned inhibition training paradigms. Exposure of
Hermissenda to explicitly unpaired (EU) presentations of
light and rotation produces persistent decreases in the excitability of
Type B cells: smaller light responses and action potential frequency
(Britton and Farley 1999), enhanced K+ currents (Farley et al. 1999
).
These inhibitory-learning correlated changes are the opposite of those
produced by light-rotation pairings. Thus PP1 is an attractive
potential candidate to play a role in these increases. A related
possibility is that PP1 mediates accelerated forgetting, caused by
extinction training in Hermissenda (Richards et al.
1984
).
One should not overlook the possibility that PP1/PP2A may directly
modulate PKC in B cells. PKC is regulated in vivo by three functionally
distinct phosphorylations (Keranen et al. 1995). Recombinant PKC
activity has been reported to be reversibly
inhibited by PP1 and PP2A (Ricciarelli and Azzi 1998
),
apparently at autophosphorylation sites. Further, at low
concentrations, PP1 has been found to activate PKC
(Ricciarelli and Azzi 1998
), implying the existence of
an inhibitory phosphorylation site. Thus the degree of PKC activity in
B cells may depend on PP1 activity, and this may introduce additional
complexities to the signaling networks that are activated during and
after conditioning. It is possible that calyculin and inhibitor-2
activated PKC in B cells and, in combination with inhibition of PP1,
stimulated phosphorylation.
PP1 inhibitor effects on light responses
Protein phosphatases are likely to affect other physiological processes in B photoreceptors that have only an incidental connection to learning, such as phototransduction.
Both membrane and soluble PP1- and PP2A-like phosphatase activity have
been shown to exist in visual and nonvisual tissue in
Limulus nervous system (Edwards et al. 1996).
The activities of both enzymes are greater in light- than in
dark-adapted lateral eyes. Furthermore, in Limulus ventral
eye photoreceptors, okadaic acid (a PP1 and PP2A inhibitor) caused a
delayed depolarization of resting membrane potential and slower and
diminished light-activated cation currents (Edwards et al.
1996
). Inhibitors of calcineurin have also been reported to
increase phosphorylation of arrestin, produce smaller and sharper
quantal bumps, and slow the activation kinetics of light-induced inward
currents in Limulus photoreceptors (Kass et
al. 1998
).
The findings from Limulus photoreceptors with PP1/PP2A inhibitors are generally consistent with our results with Hermissenda. Although the molecular phototransduction pathways have not been thoroughly determined for Hermissenda, it seems likely that phosphatase inhibitors affect the light response of invertebrate photoreceptors, and these effects occur in addition to effects on somatic K+ channels.
Limitations of present studies
A limitation of our present studies is the fact that all of our conclusions rest on pharmacological/electrophysiological evidence. Biochemical studies and characterization of phosphatases from the Hermissenda nervous system (and more specifically, Type B photoreceptors) have not yet been reported. Thus the validity of our conclusions depends heavily on the as yet untested assumptions that Type B photoreceptors express PP1 and PP2A isoforms similar to those of other eucaryotic organisms and that the Hermissenda isoforms are inhibited by the PP-inhibitors examined here with approximately the same potency and specificity as their counterparts in other organisms.
We think the likelihood that these assumptions are correct is high. The
three major S/T phosphatases families (PP1, PP2A, PP2B) are found in
the nervous systems of organisms at all phylogenetic levels, ranging
from Drosophila and Caenorhabditis elegans to rat
to humans. Mollusks that are related to Hermissenda, such as
Aplysia, also express these same phosphatases (Endo
et al. 1992). The catalytic subunits of PP1, PP2A, and PP2B are
highly conserved. Diversity appears to come about primarily through
which type(s) of regulatory subunits and accessory proteins the core catalytic subunit is associated with (Cohen 1989
;
Shenolikar 1994
; Wera and Hemmings 1996
).
Most inhibitors bind to the catalytic domain/subunit of the PPs (Herzig
and Neumann 2000
; MacKintosh and MacKintosh
1994
). The relative potency and specificity of calyculin A and
inhibitor-2 against PP1 versus PP2A and PP2B is well established and
generally accepted. The same appears to be true for cyclosporin
(PP2B-inhibitor) and cantharidic acid (PP2A).
However, it is obvious that molecular identification and biochemical characterization of PPs from the Hermissenda nervous system and photoreceptors will eventually need to be accomplished. Biochemical studies will be particularly important for answering questions like: is PP1 constitutively active in the basal state? Is learning and/or memory formation associated with a change in PP activity?
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FOOTNOTES |
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Address for reprint requests: J. Farley, Neural Science, 1101 E. 10th St., Rm. 370, Indiana University, Bloomington, IN 47405-7007 (E-mail: farleyj{at}indiana.edu).
1 In Fig. 5, the current responses to depolarization to 0 mV are normalized to the amplitudes at time 0; in Fig. 7, averages of absolute value of current amplitudes are depicted. These different ways of calculating the averages [mean of ratios (Fig. 5) vs. ratio of means (Fig. 7)] are responsible for the slight discrepancies between the two sets of figures.
Received 13 October 2000; accepted in final form 30 May 2001.
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REFERENCES |
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