1Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106; and 2Department of Physiology and Biophysics and Department of Ophthalmology, Dalhousie University, Halifax, Nova Scotia B3H 4H7 Canada
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ABSTRACT |
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Kourennyi, Dmitri E. and
Steven Barnes.
Depolarization-Induced Calcium Channel Facilitation in Rod
Photoreceptors Is Independent of G Proteins and Phosphorylation.
J. Neurophysiol. 84: 133-138, 2000.
Depolarization-induced facilitation of L-type Ca channels in
rod photoreceptors was investigated with nystatin-perforated and
ruptured whole cell patch-clamp techniques in cells isolated from tiger
salamander retina. Induction of facilitation was voltage dependent with
a half-maximal effect seen at prepulse potentials near +31 mV. Reversal
of facilitation was time dependent with fast (
20 ms)
and slow (
1 s) components at
60 mV. Incubation of
cells with pertussis toxin or intracellular administration of guanosine
5'-O-(3-thiotriphosphate) or guanosine 5'-O-(2-thiodiphosphate) had no effect on the degree to
which facilitation could be evoked, implying the absence of a
significant role for G proteins. Application of the phosphatase
inhibitor okadaic acid or inclusion of ATP, to boost levels of
phosphorylation, or inclusion of 5'adenylylimidophosphate or inhibitors
of protein kinase in the pipette, to reduce levels of phosphorylation,
had no effect on the development of facilitation, suggesting that phosphorylation has little or no role in this phenomenon. These results
show that the L-type Ca channels in rod photoreceptors, which appear to
be composed of
1F-like subunits, undergo
voltage-dependent facilitation in a manner that differs from some other
L-type Ca channels which undergo facilitation via phosphorylation or
through G-protein-mediated inhibition.
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INTRODUCTION |
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Vertebrate rod photoreceptors express
noninactivating, high-voltage-activated (HVA) calcium channels that are
considered to be of the L-type due to their dihydropyridine sensitivity
(Kurenny et al. 1994). In cones, the L-type Ca channels
have a distinct pharmacological profile (Wilkinson and Barnes
1996
), most closely resembling that of
1D-subunit-containing Ca channels
(Williams et al. 1992
), but this characterization has
not been made as completely for rods. Antibodies directed against
1D subunits labeled cones, not rods, in
mammalian retina (Morgans 1999
; Taylor and
Morgans 1998
), and it is probable that rod Ca channels are
composed of the newly identified
1F subunits
(Bech-Hansen et al. 1998
; Strom et al.
1998
). Calcium entry into rod photoreceptor terminals via noninactivating L-type Ca channels provides for the continuous release
of the neurotransmitter glutamate in darkness, and this release is
graded by light over a narrow range of hyperpolarization.
Depolarization-induced facilitation of L-type calcium channels was
first described by Fenwick et al. (1982) in bovine
chromaffin cells. Numerous studies since then have indicated, albeit
not without controversy, that it is the voltage-dependent
phosphorylation of the channels by protein kinase A (PKA) that
accounts for the phenomenon in chromaffin cells (Artalejo et al.
1990
, 1992
; but see Albillos et al. 1996
;
Doupnik and Pun 1994
), skeletal muscle (Sculptoreanu et al. 1993b
), cardiac muscle cells
(Schouten and Morad 1989
; Sculptoreanu et al.
1993a
; Tiaho et al. 1994
; but see Foley
and Pelzer 1994
), and neurons (Bourinet et al.
1994
; but see Parri and Lansman 1996
) but not in
cloned smooth muscle cells (Kleppisch et al. 1994
). In
many cases, the increase in peak Ca channel current is accompanied by a
negative shift in channel activation. For some of these L channels, it
remains an open question as to whether the channel itself or an
intermediate G protein is a target for the protein kinase (see, for
example, Dolphin 1996a
,b
; García and
Carbone 1996
). It has also been suggested that intracellular
calcium participates in facilitation either in a
phosphorylation-independent manner (Bates and Gurney
1993
) or via a calcium-calmodulin-dependent kinase pathway
(Anderson et al. 1994
; Gurney et al.
1989
).
Some neuronal Ca channels, in particular the N and P/Q
subtypes, undergo voltage-dependent facilitation that is mediated by direct interactions between -subunits of GTP-binding proteins (G
proteins) and the Ca channel (see Hille 1994
;
Zamponi and Snutch 1998
). G-protein subunits inhibit
these Ca channels and may be driven off by large depolarizations,
accounting for the facilitation. Cone photoreceptor Ca channels undergo
reversible block by
-conotoxin GVIA, invoking properties of N-type
Ca channels, but considered as well to be a property of the
incompletely characterized
1D-containing Ca
channel. In addition to conotoxin sensitivity, do photoreceptor L-type
Ca channels also share features of G-protein-mediated facilitation with
N-type channels?
Recently Kammermeier and Jones (1998) described
facilitation in L-type channels of thalamic neurons that is independent
of G proteins and phosphorylation. Similar to suggestions that direct conformational changes in the Ca channel protein underlie the mechanism
for the voltage-dependent facilitation in smooth muscle L-type Ca
channels (Kleppisch et al. 1994
), these authors
concluded that no chemical modification was necessary to produce the
facilitated state of the channels.
Since the rod photoreceptor Ca channel has not been pharmacologically
characterized and may be composed of novel 1F
subunits, we sought to characterize features of the
depolarization-induced facilitation of this L-type Ca channel. The
results presented below show that it is unlikely that the facilitation
mechanism involves phosphorylation or G proteins, suggesting that
facilitation is an intrinsic property of this Ca channel subtype.
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METHODS |
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Larval tiger salamanders, Ambystoma tigrinum, were
purchased from Kons Scientific (Germantown, WI). The animals were
stored at 4°C and then decapitated and the head hemisected or pithed. Both eyes were removed and placed in Ringer solution containing (in
mM): 90 NaCl, 2.5 KCl, 3 CaCl2, 15 HEPES 15, and
10 D-glucose (pH 7.6). After dissection of the
cornea, iris, and lens, the retina was peeled gently from the eyecup
and mechanically triturated. The dissociated cells were allowed to
settle in a 0.25-ml recording chamber. Whole cell recordings were
obtained form the inner segment of intact rod photoreceptors using both
ruptured- and perforated-patch techniques. All experiments were
performed under room light and microscope illumination (1-2
Wm2) and at room
temperature (20-23°C).
Silicone-elastomer (Sylgard)-coated fire-polished patch pipettes
were pulled on a Kopf puller (model 730, Tujunga, CA) from hematocrit
glass tubes (VWR, West Chester, PA) and filled with a solution
containing (in mM): 95 CsCl, 3 MgCl2, 10 HEPES,
and 1 EGTA (pH 7.2). Drugs were included in the pipette solution, during rupture-patch recording, or added to the superfusing solution. For perforated-patch recordings, nystatin was dissolved in DMSO and
included in the pipette solution (150 µg/ml). Three superfusing solutions were modified from the Ringer solution described in the
preceding text to contain (in mM): 5 BaCl2, 50 TEACl, and 15 CsCl; 10 BaCl2, 50 TEACl, or 10 CsCl; or 10 BaCl2, 25 TEACl, and 10 CsCl; where
for each solution, BaCl2 replaced
CaCl2 in an equimolar manner and TEACl and CsCl
replaced equimolar NaCl. The concentration of HEPES was increased to 20 mM. In each series of experiments used for statistical analysis, the
ionic conditions were identical.
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (Iso-H-7),
5'-adenylylimidophosphate (AMP-PNP), guanosine
5'-O-(3-thiotriphosphate) (GTP- -S), guanosine
5'-O-(2-thiodiphosphate) (GDP-
-S), adenosine 5'-O-(2-thiodiphosphate) (ADP-
-S), and ATP were obtained
from Sigma (St. Louis, MO). Okadaic acid was obtained from LC Services Corp. (Woburn, MA) and RBI (Natick, MA). Bay K 8644 was obtained from
ICN Biochemicals (Cleveland, OH). PKI 5-24 was obtained from Peninsula
Laboratories, (Belmont, CA).
Currents were recorded using an Axopatch-1B amplifier, TL-1 interface,
and BASIC-FASTLAB software running on a 386 computer. Facilitation was
induced by a depolarizing prepulse to 100 or 120 mV for 50 ms. Membrane
currents were averaged over the last 2-5 ms of 25-ms voltage steps
from a holding potential of 60 or
70 mV. We measured currents near
the end of the test pulse to minimize possible current artifacts
induced by the capacitive discharge following the large
facilitation-inducing steps to +100 or +120 mV. However, since
facilitation is removed at negative potentials with a bi-exponential
time course having fast (
20 ms) and slow (
1 s) components, it is likely that such measurement near
the end of the test pulse underestimates the degree to which
facilitation occurred since some of the effect would have decayed
during the first 20-23 ms of the test pulse. Activation curves were
constructed from series resistance and leak corrected I-V
relationships after division by the driving force. Leak correction was
performed by subtracting a line fit to the I-V relation near
60 mV (typically from
70 to
50 mV). Activation curves were then
constructed by dividing the I-V relation by a line fit to
the relatively linear portion of the I-V relation just
positive to 0 mV. This approach treats the voltage range over which Ca
channels activate (usually between
30 and 0 mV) as if there were a
linear driving force, a prudent approximation that yields a valid
description of channel activation, but one that assigns an artifactual
reversal potential substantially more negative than the values observed
or expected for nonlinear, fully-activated Ca channel currents (see
Fig. 1B for example). The
Boltzmann equation (g = gmax/{1 + exp[(V0.5
V)/s]}) was used to fit activation curves,
where g is a conductance due to calcium channels estimated from the slope of the fully activated, linear part of the
I-V relationship, V0.5 is
the half-activation potential, and s is the slope factor.
Statistical data are presented as means ± SE. The absence of
error bars in some graphs indicates that either only one cell was
viable at the time of recording or that the error bars are smaller than
the symbols. Statistical significance of facilitation was estimated
using paired one-tailed Student's t-test unless otherwise
indicated.
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RESULTS |
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L-type calcium channels are present in photoreceptors
Figure 1 shows typical rod photoreceptor Ca channel barium
currents recorded using perforated-patch whole cell techniques. Ca
channel currents were activated at potentials positive to 40 mV and
reached a maximum amplitude at about
10 mV. Application of
nifedipine, a blocker of L-type channels, suppressed the Ca channel
current (Fig. 1D), while Bay K 8644, an L-type channel agonist, caused a dramatic increase in current magnitude (Fig. 1E). The blocker of N-type calcium channels,
-conotoxin
GVIA, generally produced a slight decrease in Ca channel current,
although in some cells no effect was seen (Fig. 1D). These
data, taken together with the noninactivating nature and high
activation threshold of Ca channel current, indicate that rods express
an L-type calcium channel. It should be noted that these properties of
calcium channels in rod photoreceptors appear generally similar to
those in cone photoreceptors of this same species, although the block
by
-conotoxin GVIA is greater in cones (Wilkinson and Barnes
1996
).
Facilitation of Ca channel current
Ca channel current could be increased, or facilitated, by
application of a strong depolarizing voltage prepulse (Fig.
2). The average increase in the maximum
conductance after a prepulse to 100 mV was 22.7 ± 5.9%
(n = 16, 0.001 < P < 0.01) in
perforated-patch recordings and 21.0 ± 4.6% (n = 13, P < 0.001) in ruptured-patch recordings. No
significant change in the half activation potential was observed in
either case: V0.5 was 0.60 ± 0.46 mV (n = 16, 0.2 < P < 0.3)
in perforated-patch recordings and 0.61 ± 0.66 mV
(n = 13, 0.3 < P < 0.4) in
ruptured-patch recordings. The slope factor also did not change
significantly (increase of 2.3 ± 1.9%, 0.2 < P < 0.3 and 7.0 ± 2.5%, 0.01 < P < 0.02, respectively). We also calculated the
percentage increase in peak Ca channel current, measured at a potential
usually between 20 and 0 mV, to be 35.4 ± 5.6%
(n = 30, P < 0.0005) in
perforated-patch and 41.5 ± 13.8% (n = 7, 0.01 < P < 0.025) in ruptured-patch recordings.
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Facilitation is voltage dependent
The extent to which Ca channel current was facilitated depended on conditioning depolarization. Such a dependence could be described well by a Boltzmann function (Fig. 3A). In three cells recorded from with the permeabilized patch technique, the prepulse amplitude causing 50% of the maximum facilitation amount was +30.5 ± 16.3 mV and the slope factor was +26.5 ± 6.0 mV.
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Time-dependent removal of facilitation
The facilitation of Ca channel current was gradually eliminated with an increase in the gap period between a prepulse and a test pulse (Fig. 3B). In the same cells, described in the preceding text, in which the voltage-dependence of facilitation was established, the time-dependent process appeared to be double exponential with time constants of 22 ± 6 and 835 ± 400 ms (n = 3). Note the large standard error in some of these values.
L-type calcium channels are facilitated
Application of the nonselective calcium channel blocker, cadmium,
eliminated Ca channel current and the facilitated component to an
extent that quantitative analysis was impossible (Fig.
4A). The L-type channel
blocker nifedipine (1 µM) suppressed Ca channel current dramatically
and made facilitation less significant than in control (Ca channel
current was increased after the prepulse by 34.9 ± 15.5%,
n = 5, 0.05 < P < 0.1, see Fig.
4B). The L-type channel agonist Bay K 8644 (0.5-1 µM)
increased Ca channel current substantially and did not affect the
degree of facilitation (21.9 ± 4.2%, n = 8, 0.001 < P < 0.005, see Fig. 4C). The
effects of -conotoxin GVIA were minor on both the Ca channel current
and the degree of facilitation (39.1 ± 12.2%, n = 4, 0.02 < P < 0.05, see Fig. 4D).
These results show that it was L-type calcium channels that were
facilitated in rod photoreceptors by depolarization.
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G proteins are not involved
One well-defined intracellular mechanisms by which calcium
channels can be facilitated in a voltage-dependent manner is through the unbinding of G proteins from the channels. While this mechanism has
been demonstrated most clearly in the case of N-type Ca channels (for
reviews, see Hille 1994; Zamponi and Snutch
1998
), we sought to test a role for G proteins in the
facilitation of rod L channels.
As described in the preceding text, facilitation occurred equally with perforated- and ruptured-patch recordings, casting doubt on whether any soluble messengers have a role in this phenomenon (Fig. 5). However, many intracellular processes, in particular some involving G proteins and phosphorylation, are membrane delimited. Therefore we used pertussis toxin (PTX) to block membrane delimited PTX-sensitive G proteins. Figure 5 shows that facilitation still occurred in rods dissociated from retinas pretreated with 5 µg/ml PTX for 4 h. On average, with perforated-patch recordings from cells treated with 0.3 µg/ml PTX for 1-2 days, 3 µg/ml PTX overnight, or 5 µg/ml PTX for 4 h, Ca channel current was increased after the prepulse by 41.2 ± 8.0% (n = 12, P < 0.0005).
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The failure of PTX to block facilitation suggested that this effect was
independent of PTX-sensitive G proteins. To further address the issue
of the G-protein involvement in facilitation, we activated G proteins
with 1 mM GTP--S or locked G proteins in their inactive form using 2 mM GDP-
-S in the pipette solution. In both cases, the facilitation
persisted, Ca channel current being increased by 23.8 ± 1.5%
(n = 2, 0.01 < P < 0.025) and
37.8 ± 14.6% (n = 6, 0.01 < P < 0.025), respectively, suggesting a G-protein-independent mechanism (Fig. 5). The amount of facilitation seen with GTP-
-S in the pipette was not significantly different from
control (0.8 < P < 0.9; Student's unpaired
t-test).
Phosphorylation is not involved
Phosphorylation-dependent facilitation has been shown for L-type
calcium channels in several preparations (Artalejo et al. 1990,
1992
; but see Albillos et al. 1996
;
Doupnik and Pun 1994
), skeletal muscle
(Sculptoreanu et al. 1993b
), cardiac muscle cells (Schouten and Morad 1989
; Sculptoreanu et al.
1993a
; Tiaho et al. 1994
; but see Foley
and Pelzer 1994
), and neurons (Bourinet et al.
1994
). We checked this possibility in rod photoreceptors with
special interest since earlier work had shown that another type of rod
ion channel, a voltage-gated potassium channel, was regulated by
phosphorylation (Kurennyi and Barnes 1997
).
Figure 6 summarizes the results of a battery of tests designed to define the role of phosphorylation. When phosphorylation was favored and dephosphorylation suppressed by extracellular application of 0.5-1 µM okadaic acid in perforated-patch recordings, depolarizations still increased the maximum conductance reversibly by 33.6 ± 11.4% (n = 8, 0.02 < P < 0.025), an amount statistically indistinguishable from that in control (0.3 < P < 0.4, unpaired t-test). There was no significant shift in the half activation potential (V0.5 = 0.11 ± 0.70 mV, 0.8 < P < 0.9). In ruptured-patch mode, the inclusion of 1 µM okadaic acid together with 1.8 mM ATP in the pipette (n = 3) also resulted in facilitation similar to that seen during control ruptured-patch experiments (maximum conductance increase of 27.7 ± 5.6%, 0.02 < P < 0.05; V0.5 = 0.21 ± 0.28 mV, 0.5 < P < 0.6).
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An additional approach taken to address the problem was to suppress
phosphorylation. We included in the patch pipette either 100 µM H-7
(a concentration that would likely block both cyclic nucleotide
dependent kinases and PKC, n = 2), 1 µM PKI 5-24
(PKA inhibitor) with (n = 4) and without 1 mM ATP
(n = 3), 0.5 mM AMP-PNP (nonmetabolizable analog of
ATP, n = 14), or 2 mM ADP--S (to suppress adenylyl
cyclase, n = 2) in ruptured-patch experiments. In all
cases, facilitation persisted at the control level (the maximum
conductance increased by 47.1 ± 39.5, 24.7 ± 9.2, 25.6 ± 5.4, 43.7 ± 9.8, and 28.9 ± 13.8%, respectively).
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DISCUSSION |
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We interpret these findings to indicate that neither
G-protein-coupled nor phosphorylation-dependent mechanisms are involved in voltage-dependent facilitation of L-type calcium channels in rod
photoreceptors. It remains a possibility that G-protein- or phosphorylation-dependent mechanisms, unaffected by the drugs used
presently, mediate aspects of the process of facilitation or its
removal. Since none of our manipulations of the
intracellular milieu affected facilitation, we further doubt a role for
a soluble messenger system. We suggest that the likely mechanism
responsible for facilitation may be via a direct voltage-dependent
conformational change of the channels, similar to that suggested for
L-channels in smooth muscle and thalamic neurons (Kammermeier
and Jones 1998; Kleppisch et al. 1994
).
L-type calcium channels are represented by a number of different
1 subunits:
1S
(skeletal muscle),
1C (cardiac, smooth muscle,
brain),
1D (kidney, brain) (reviewed in
Dunlap et al. 1995
), and
1F
(rod photoreceptors) (Bech-Hansen et al. 1998
; Strom et al. 1998
). Calcium channels in both salamander
cones (Wilkinson and Barnes 1996
) and rods (present
study) share a similar pharmacological profile, e.g., sensitivity to
both dihydropyridines and
-conotoxin GVIA, with
1D L-type Ca channels, suggesting that this
may be characteristic of
1F Ca channels. The
voltage-dependent facilitation of putative
1D
channels in cerebellar granule cells (Parri and Lansman
1996
) has properties consistent with those of rod photoreceptors.
The molecular structures responsible for G-protein-mediated,
voltage-dependent facilitation of 1A and
1B channels (P/Q and N types, respectively),
as well as some
1E channels (possibly R type)
offer speculations into the problem of G-protein-independent facilitation. For these channel types, the domain I-II cytoplasmic linker has been identified as the protein region responsible for binding G-protein subunits (reviewed in Zamponi and Snutch
1998
). Furthermore binding of syntaxin to synaptic N-type
channels modulates G-protein-dependent facilitation (Stanley and
Mirotznik 1997
). It may be expected that analogous cytoplasmic
regions of the G-protein-independent channels, such as those in rods,
impart equivalent channel modulatory function in the absence of
cytoplasmic molecules such as G proteins or syntaxin, in a manner
independent of protein phosphorylation.
The molecular mechanisms responsible for L-channel facilitation in rod
photoreceptors remain unclear, and a solution to this problem will
likely require combined electrophysiological, biochemical, and
molecular biological approaches. The present results offer evidence
that the L-type Ca channels in photoreceptors are unique with respect
to the facilitatory properties of 1A-,
1B-,
1C-, and
1E-like Ca channels. This finding furthers the
functional distinctions being drawn between the classes of L-type Ca
channels (e.g.,
1F L-type Ca channels as
compared with
1C).
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ACKNOWLEDGMENTS |
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This work was supported by the Medical Research Council (MRC) of Canada and the Johansen Research Fund of the Fight for Sight research division of Prevent Blindness America.
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FOOTNOTES |
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Address reprint requests to: S. Barnes, Dept. of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada (E-mail:sbarnes{at}is.dal.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 December 1999; accepted in final form 16 March 2000.
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REFERENCES |
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