Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2+ and trp

cell Ca/ciurn (1995) 18. 256-274 0 Pearson Professional Lld 1995

Phosphoinositide-mediated Drosophila photoreceptors: RP

phototransduction in the role of Ca*+ and

R.C. HARDIE’ and El. MINKE2 ’ Department of Anatomy, University of Cambridge, Cambridge, UK and *Department of Physiology, Hebrew University Hadassah Medical School and the Minerva Center for Studies of Visual Transduction, Jerusalem, Israel Abstract - Drosophila photoreceptors represent a paradigm for the genetic dissection of phototransduction and, more generally for Ca*+ signalling. As in most invertebrates, phototransduction in Drosophila is mediated by the phosphoinositide (PI) cascade and is completely blocked by null mutations of the norpA gene which encodes a phospholipase C-p isoform. The light-activated conductance in Drosophila is normally highly permeable to Ca*+ but in null mutants of the trp gene Ca*+ permeability is great1 reduced. Furthermore, the kp gene sequence shows homologies with voltage gated Ca Y+ channels, suggesting that trp encodes a light-sensitive channel subunit. Ca*+ influx via these channels is instrumental in light adaptation, and profoundly influences phototransduction via positive and negative feedback at multiple molecular targets including protein kinase C. The mechanism of activation of the light-sensitive channels remains unresolved. A requirement for Ca*+ release from internal stores is suggested by the finding that Drosophila photoreceptors cannot sustain a maintained response under various conditions which might be ex ected to result in depletion of Ca*+ stores. However, Ca*+ release cannot be detected by Ca !if+ indicator dyes and raising Ca2+ by photorelease of caged Ca*+ fails to mimic excitation. Recent studies, both in situ and with heterologously expressed frp protein, suggest that the frpdependent channels may be activated by a process analogous to ‘capacitative Ca*+ entry’, a widespread, but poorly understood mode of PI-regulated Ca*+ influx in vertebrate cells.

Vertebrate compared

and invertebrate

phototransduction

It has long been recognized that phototransduction in both vertebrates and invertebrates belongs to the general class of G-protein coupled signal transduc-

tion which is a virtually ubiquitous feature of all cells. Common to all G-protein coupled signaling is the interaction of a membrane-bound receptor, with a heterotrimeric G protein. The major diversity is found in the nature of the receptor molecule and effector enzyme systems, the most familiar of which 256

PI-MEDIATED

PHOTOTRANSDUCTION:

& TRP GENE

Ca IONS

bumps

flash 20 pA 20 ms

L Fig.

1 Quantum

dim

light

train

bumps

delivered

of discrete

in Dlnsophiko

to a voltage inward

currents,

The response

absorbed

photon

of light.

containing

ca

100 photons

sponse Since

is the linear the

latency.

the flash

waveform their single

bumps

contributing response

(dotted

rising wild

type

photoreceptor

the response to a flash At

this

of light intensity,

were using

made

have

the re-

the dissociated

[49].

a variable

than the bump

bumps, at -70

a

(arrow)

bumps

broader

of IO quantum

elicits to a single

of the contributing

is considerably

Recordings

A step of

photoreceptor

to the response

line. average

phases).

each

is shown.

summation

photoreceptors.

clamped

aligned mV

from

by a

ommatidial

preparation.

include the adenylate cyclase, phosphodiesterase and phospholipase C signaling cascades [l-3]. Vertebrate and invertebrate phototransduction share homologies at the level of receptor (rhodopsin) activation and inactivation [4,5]; however, it appears that the downstream effector systems are distinct. Thus, whilst the vertebrate G-protein, transducin (Gi), activates a phosphodiesterase catalyzing the hydrolysis of cGMP [2], evidence detailed below indicates that the key effector enzyme in most invertebrate photoreceptors is phospholipase C, responsible for generation of the soluble messenger, myo-inositol 1,4,5 trisphosphate (InsP3) which plays a central role in cellular Ca*+ signaling [3,6]. Invertebrate photoreceptors also have a characteristic structure quite distinct from the stacked disc arrangement of the vertebrate rod. The light-sensi-

257

tive membrane, containing rhodopsin, is composed of tightly packed microvilli along the length of the cell, typically forming a long light-guiding structure known as the rhabdomere. Immuno-gold labeling has revealed that much of the transduction machinery in invertebrates, including G-protein [7] and phospholipase C [8] is localized in the microvilli. A second important compartment is formed by a system of submicrovillar cisternae (SMC), which is a specialized subcompartment of the smooth endoplasmic reticulum [9] closely abutting the base of the microvilli and which is believed to represent InsP3sensitive Ca*+ stores [lo]. Both vertebrate and invertebrate photoreceptors achieve the ultimate in sensitivity, responding to individual photons with discrete electrical events, quantum bumps ([ 1 I]; Fig. 1) which sum to form the macroscopic response ([ 121, but see [ 131). In vertebrate photoreceptors quantum bumps show little variation in latency, consequently the macroscopic response to a dim flash in the linear range simply resembles a scaled-up bump [ 141. By contrast, quantum bumps in invertebrates are characterised by a variable latency followed by an abrupt rising phase. The macroscopic response to a flash, which is generated by the convolution of bump shape and latency distribution, is thus significantly broader than the single bump ([15]; Fig. 1). Since the amplitude and duration of bumps are uncorrelated with their latency (Limufus [11,12,15-171; locust [18]), it is suggested that different processes with low gain (-5-10) determine the latency while other, high gain processes characterised by positive and negative feedback loops, determine bump shape. Comparison of the latency to light and direct InsP3 injection suggest that most of the latency period may be determined by stages up to InsP3 production, whilst the bump shape and amplitude are determined later on in the cascade [19]. It has been widely accepted that modulation of bump shape, amplitude and latency dispersion are major determinants of the gain and frequency response of the receptor potential during light adaptation [ 12,151. As cells highly specialized for phosphoinositide (PI) signaling, invertebrate photoreceptors represent excellent models for studies of this ubiquitous Ca*+ signaling pathway. This is particularly true in Drosophila which has the unique advantage of a mole-

CELL

258

cular genetic potential unparalleled amongst higher animals. This review, after first briefly summarizing the evidence for the involvement of the PI cascade in invertebrate phototransduction, concentrates on the functional and molecular nature of the light-sensitive channels in Drosophila and then addresses one of the major unresolved questions of both phototransduction and Ca*+ signaling generally: namely how is activation of the PI pathway linked to the opening of the plasma membrane channels? The discussion will draw largely upon recent studies from Drosophila. Although studies from other invertebrate preparations will also be discussed where relevant, the reader should be aware that there are quantitative, and possibly qualitative, differences in the detailed mechanisms of phototransduction among various invertebrate species.

Phototransduction inositide pathway

is mediated

by the phospho-

A large body of biochemical evidence indicates that light activates a phosphoinositide cascade in invertebrate photoreceptors (Fig. 2; fly [20-221; Limulus [23]; squid [24-261). Pharmacological experiments in intact photoreceptors of Lirnufus and fly also indicated that compounds which are known to activate the phosphoinositide cascade, excite and adapt the photoreceptors cells (GTPyS: Lirnulus [27,28]; fly [29,30] - InsP3: Limulus [23] fly [20,30]). Conversely, pharmacological agents which inhibit the PI pathway are found to block the light response. Thus, polyamines, like neomycin and spermine, which inhibit the interaction of PLC with its substrate phosphatidylinositol 4,5 bis-phosphate (PIPz; [32]) reduce a large fraction of the light response of Limulus ventral photoreceptors [33,34] as does heparin, which is a (non-specific) antagonist of the InsP3 receptor [33]. Further strong evidence that the phosphoinositide cascade is necessary for excitation has come from the isolation and analysis of specific Drosophila transduction mutants. The most compelling evidence derives from the ‘no receptor potential A’ (norpA) gene which encodes a phospholipase C-p isoform associated with the microvillar membrane [8,35]. In

CALCIUM

null and temperature-sensitive norpA mutants, lightactivated PLC activity in eye membrane preparation is inhibited [21,22,36]), whilst the light response is largely (electroretinogram [37]) or completely abolished (intracellular recordings [38]; whole cell recordings [39,40]). Weaker norpA alleles increase bump latency and dispersion without affecting bump shape [41] supporting the proposal that the quantum bump shape is determined downstream from PLC. More generally, it is striking that all of the ca 20 Drosophila gene products shown by mutational analysis to be involved in phototransduction, in as far as they can be assigned to any specific G-protein coupled effector system, are components of the PI cascade (Table, Fig. 2). In addition to norpA, these include the PLC-/3 specific G-protein, Gq [7,42], the related Gp subunit [43,44] genes involved in PI turnover including DAG kinase (rdgA [45]), a putative phosphatidyl inositol transfer protein (rdgB [46]) and CDP-diacylglycerol synthase (an enzyme required for PIP2 regeneration [47]), as well as a Ca*+ dependent protein kinase C (inaC [48]). By contrast, whilst some important transduction genes have proven to represent novel gene families, none have been found to be associated with alternative Gprotein coupled signaling pathways, such as those involved in cyclic nucleotide metabolism. In short, the above studies have provided strong evidence: (i) that a light-activated PI cascade of high capacity operates in invertebrate photoreceptors; (ii) that activity of the cascade (i.e. InsP3 production) is s@cient for excitation; and (iii) that this cascade (as defined by the presence of functional, light-activatable phospholipase C) is required for excitation. Despite this body of evidence, the mechanism by which the PI cascade leads to the final response, namely the opening of cation selective ion channels in the plasma membrane, remains a major unresolved question. Significant advances have been made however, in the functional and molecular analysis of the light-sensitive channels - information that may ultimately provide vital clues to their mechanism of activation. In the following, we first describe some of the properties of the light-sensitive conductance in Drosophila photoreceptors, in particular summarizing evidence implicating the transient receptor potential (trp) gene product as an integral channel subunit gene. Secondly, we consider

PI-MEDIATED

PHOTOTRANSDUCTION:

Ca IONS

Pigment

& TRP GENE

cycle

259

b,ue EXCITATION

,

rdgA

Fig.

2 Phototransduction

forrnlng transduction

cascade

(see also turnover

Table).

are shown

The

acti\,ates

cycle)

major

which

phospholipase

binds

product) I@-sensitive feedhack

(IPiR)

in the plasma

channel. at several

r~oj.pA).

Notice

target

on absorption

of a blue photon.

(Mpp)

and binding

to one and possibly

to R resulting

in dissociation

Arrestin

(also

known

phatidic

acid (PA)

then to phosphatidyi membrane.

Two

as phosrestin)

by DAG inositol kinases

kinase (PI).

complete

(Rh)

In addition

role of Ca”:

itself

of arrestin

is phosphorylated PI transporter

the resynthesis

a substantial

and together to active

fraction

After

PA is converted

dependent to CDP-DAG

of a second

M* which

absorption

by R phosphatase

in a Ca CaM

manner.

is inactivated (red)

PI turnover:

DAG

synthase PI from

-

and InsP3.

of Nat

(trl,

gene

permeable

and mediates

gene product).

gene product)

via CDP-DAG

see text and Table.

(DAG) channels

class

M*

the G, a subunit

conductance

(innC

of a second

(r&C

by the r-d,gB gene may transfer

of PIP2. For references

(metarhodopsin

for GTP

diacylglycerol

PKC

in italics

and phosphoinositide

Ca2+ permeable

activates

of the

are shown

state,

of the light-activated

DAG

metarhodopsin

(37 and 49 kD).

encoded

to generate

microvilli

components

cycle

of GDP

Subsequently.

for the existence with

pigment

to the active

with

Major

components

and exchange

the SMC.

is evidence

dephosphorylated

(r.1$+4 gene product); A putative

IPqR

(R) is converted

R is finally

from

there

it mediates

the channels, two forms

Ca”

the various

of the visual

On dissociation

PIP2 (phosphatidyl45bisphosphate)

of a photoreceptor

Ca*+ stores.

is converted

releasing

rhodopsin

of arrestin.

details

rhodopsin

G, protein.

encoding

presumably

including

cycle:

Genes

on the right:

cross-section

the putative

PLC cleaves

membrane

the pivotal

sites,

a schematic

represent

microvillus.

of blue light.

the heterotrimeric

shows

(SMC)

is shown

absorption

with

right)

cistemae

of excitation

followmg

receptor

(upper

the base of a single

pathway

C (PLC.

Inset

of submicrovillar

around

interacts

to the InsP?

are activated

in D~so@i/n.

A system

on the left. Excitation:

qee pigment InsPi

cascade

the rhabdomere.

Pigment

by phosphorylation photon,

M reconverts

to complete is converted

the cycle. to phos-

(cds gene product),

the SMC

to the microvillar

and

260

CELL

some implications of these and other results for the still unresolved mechanism of excitation. Properties of the light-sensitive conductance Detailed analysis of the light-sensitive conductance in Drosophila has been made possible by the recent development of a preparation of dissociated ommatiTable

Mutants

and genes

genes for which mutants involved in inactivation; potentially transduction,

involved

in phototransduction

are available: (c) ‘housekeeping’

CALCIUM

dia in which high resolution whole-cell voltage clamp recordings can be made from individual photoreceptors using patch clamp electrodes [49,50]. The preparation has also been adapted for simultaneous whole-cell recording and Ca*+ fluorimetry using fluorescent Ca*+ sensitive indicator dyes (Fig. 3 [39,40,51]), as well as for flash photolysis of caged compounds [40].

in Drosophila.

(a) genes

These are arranged as follows: (a-c) cloned in direct route of excitation; (b) those in PI metabolism; (d) cloned genes

encoding proteins involved proteins involved, for example,

involved in transduction but for which but which have not yet been cloned.

mutants

are not yet available;

and (e) mutants

Gene

Mt1p

flill~~E

92B8-

RI -6 opsin

no rhodopsin

4v

49B

G,a-subunit

overexpression

X&e

76CI

G P-subunit

100x sensitivity

WrpA

4B6-C

PLC

blind

trp

96C5-6

in&

53E6- 10

017 (37)

36Dl-2

nrr (49)

66D

49kD

very

slow

IllOD

59B l-4

novel

slow

termination

nirruc

28Al-3

myosin

rdgA

8CI

DAG

rd,qB

12CI

PI transporter

rdgC

77BI

cd.t

668

rrinnA

2lD

Protein

Mulnnt

in genes

phenotype

affecting

Reference

(a) II

I

light sensitive

Ca channel

[ll2,113] affects

excitation

response

decay

PKC

response

inactivation

37 kD arrcstin

enhances

arr(49)

VA21 [431 [351

loss

(see text)

WI

(b)

arrestin

termination

multiple

kinase

(see text)

phenotype

[481 [114,115] [116,117]

F331

[ttsl

effects

(0

kinase

light independent

degeneration

light dependent

degeneration

Rh phosphatase

light dependent

degeneration

CDP-DAG

synthase

response

decay

rhodopsin

chaperone reduced

rhodopsin

cyclophilin

homologue

[451 1461 WL841 [471 [II91

(d) R/12-4

various

R7/ocellar

trpl

46A

cation

dip

83A5-9

irwA

no mutant

[120-1221

no mutant

WI

lPs receptor

no mutant

[123,124]

2-44

not cloned

inactivation,

no afterpotential

11251

inaB

2SC-28B

not cloned

inactivation,

no afterpotential

rrinnB

87ESFl2

not cloned

reduced

[I251 u251

flillllD

3681-3

not cloned

reduced

rhodopsin

[I251

not cloned

reduced

response

unpublished

opsins

channel

(e)

rrpA -D

rhodopsin

PI-MEDIATED

PHOTOTRANSDUCTION:

Ca IONS

101lic selectivio

The ionic selectivity of the light-sensitive conductante has been estimated from ionic substitution studies [49,50,52]. Although the conductance is permeable to monovalent cations (including large organic cations such as Tris and TEA), the strong dependence of reversal potential (Erev) on extracellular Ca2+ indicates a high permeability for Ca*+ (Pc~:PN~ estimated as ca 4O:l on constant field assumptions

a)

WILD

261

& TRP GENE

]52]). A massive light-induced Ca*’ influx has also been directly demonstrated using Ca2+ indicator dyes and Ca*+ selective microelectrodes. (Fig. 3 [39,5 1,531). The demonstration that the light-sensitive conductance constitutes a Ca2’ influx pathway raises the interesting possibility that excitation in Drosophila may represent an example of the widespread, but poorly understood, phenomenon of phosphoinositide regulated Ca*’ influx - the so-called ‘capacitative Ca entry’ mechanism [M-56].

TYPE

trp

-l-.-J-P5x104 ..--.

CPS

1,-l; -. I

1

/--

PA L/ 200 ms

b) -

trP

EGTA

fP

k2

10

200 rns

1.5

Is&

clw-li---r --I7

mM

Ca

I-------

Eta

7

mV

-L+----

1 min

Fig. 3 Light-induced Ca*+ Influx in Drosrrp/ri/n photoreceptors is blocked by removing external Ca2+ and reduced by the trp mutation. (a) Calcium Green-5N (100 uM) fluorescence (upper) and light-induced current (LIC, bottom) measured simultaneously in adult (~4 h) wild type Drosoplriln photoreceptors (left) and in the rrp mutant (right) at -50 mV holding potential. The rrp mutation largely (-3-fold in average) reduced the Ca2+ stgnal relative to wild type. The dotted line indicates the resting Ca2+ level in the photoreceptor. (b) Simultaneous measurements of Flu+3 fluorescence (500 pM, upper trace) and LIC (lower trace) in wild type photoreceptors voltage clamped at -50 mV, 6 min after Ringer’s solution containing no added Ca2’ and 0.2 mM EGTA was applied. The bottom traces show similar measurements obtained 3 min after I .S mM Ca*+ was added to the Ringer’s solution. (c) Light-induced [Ca*‘] changes in the retinal extracellular space of wild type (WT. left) and rrp mutant of Drosophila (right) recorded from intact eyes under identical conditions using Ca-selective microelectrodes. The top traces are the extracellularly recorded receptor potentials (field potential,fi). The middle row is the light-induced potentiometric potential changes (A&J of the calcium-selective barrel of the Ca*+ microelectrode. The bottom traces give the time scale and the light monitor (LM). The figure shows that AE&, is largely reduced in rrp relative to WT indicating reduced Ca*’ influx. From [39.53].

262

CELL CALCIUM

of the

The effect

trp mutation

The try mutant of Drosophila [57,58] and the no steady state (nss) mutant of the sheep blowfly Lucilicl [30,59,60] are so-called because the response decays to baseline during prolonged light stimuli of medium intensity (Fig. 4). Due to the ability of LX’+, a blocker of Ca2+ channels and transporters, to mimic the trp [61] and nss [62] phenotype in wild type (WT) flies, it has been suggested that the trp gene product (TRP) is a Ca2+ transporter which refills the InsPs-sensitive Ca2+ stores during light and which is activated by a capacitative Ca*+ entry mechanism ([63], see below). The role of TRP as a Ca2+ transporter or channel has been strongly supported by measurements of the reversal potential of the light-activated conductance in trp mutants. In trp. the reversal potential is considerably more negative than in WT and also shows a much reduced dependence on extracellular Ca2+, thus indicating that the relative Ca*+ permeability is reduced ca IO-fold. This behaviour is again precisely mimicked in WT

a)

WT

S s 5 5 i

WT:

tw

-

-

-

-

0 Co”

-

pupa -

_--

IO 20

‘1 it

-10

yfs

/ , .005 .o 1

. .1...,1

* . .8..11,

.l

1

externel

[Ca”]

mM

by extracellular application of IO-20 pM La3+ (Fig. 4, [52]). The critical role of TRP in Ca*+ influx was directly demonstrated by recent measurements of Ca*+ influx in WT and trp, using fluorescent Ca2+ Indicators in dissociated ommatidia (Fig. 3a, [39]), and Ca*+-selective micro-electrodes in intact animals, (Fig. 3c, [53]). These findings strongly support the hypothesis that TRP provides the main route for Ca2+ entry into the cell during light. Independent evidence for an effect of the trp mutation on the light-sensitive channels comes from analysis of the effects of Mg*+ on the light sensitive current [64]. In common with many Ca*+ and nonselective cation channels, the light sensitive conductance is partially blocked by Mg*+. In the absence of extracellular Ca*+, ca 95% of the light sensitive current is blocked by physiological concentrations (4 mM) of Mg*+ with a Kt12 of ca 280 PM Mg2+. The Mg*+ block is strongly voltage-dependent, being relieved at both hyperpoltised and depolarised potentials. This indicates that Mg*+ blocks the pore of the channel and also that with sufficient

I .

I’“? 10

Fig. 4 The rrp phenotype. (a) In response to prolonged bright illumination, the voltage clamped response of a WT photoreceptor is characterised by a peak response and a plateau which is maintained indefinitely. The rapid peak-plateau transition is a manifestation of light adaptation and is mediated by a Ca*+-dependent reduction in the gain of transduction. In response to an identical stimulus, the response in a rrp photoreceptor rapidly decays to baseline, in this case due to exhaustion of excitation [99]. A similar decay can be elicited in WT photoreceptors under a variety of conditions where Ca2+ may be expected to be limiting: these include application of La3+ (not shown), prolonged exposure to 0 Ca*+ Ringer (WT: 0 Ca) and also a critical pupal stage (pupa). For further details see text. Adapted from [52,100]. (b) The primary defect in the trp mutant is revealed as an alteration in the ionic selectivity of the light-activated conductance. Measurements of reversal potential (E,“) in WT photoreceptors show a strong dependence on external Ca*+. In rrp, Env shows a reduced dependence on Ca2+. On constant field assumptions the relative permeability for Ca2+ with respect to monovalent ions (Na or Cs) is ca 4O:l in WT but only 3.5:l in rrp. The inset shows current-voltage relationships of the light-activated conductance in WT and rrp, both determined in Ca” free Ringer. The ca IO mV shift in E,” (arrows) is clearly visible. The S-shaped rectitication around reversal potential in WT (but not rrp) is caused by a voltage-dependent Mg*+ block. Adapted from [52].

PI-MEDIATED

PHOTOTRANSDUCTION:

Ca IONS

hyperpolarization Mg2+ permeates the channels as previously concluded from measurements of Erev. Strikingly, the Mg2+ block is greatly reduced in the trp mutant (only ca 50% block, K1/2 4 mM Mg*+), again indicating that the trp gene is involved in determining the physical structure of the pore of at least one class of light-sensitive channel. trp encodes srrDw7it

a

putative

light-sensitive

263

& TRP GENE

channel

The effects of the trp mutation on the biophysical properties of the light-sensitive conductance would appear to allow two interpretations: either the pore of the light-sensitive channel is altered in the trp mutant, or there are at least two classes of light-sensitive channel, one with the permeation and block properties found in the trp mutant and one, with high Ca*+ permeability and a high affinity Mg2+ binding site, which is absent in the trp mutant. The ability of La”+ to mimic the effect of the trp mutation on the reversal potential supports the latter hypothesis. However, since the trp gene encodes a putative channel subunit of a multimeric channel (see below) it might also be hypothesized that the native channel is a heteromultimeric assembly and that, in the trp mutant, the channels are assembled from a different subunit complement, resulting in altered pore properties. In this case, one would also need to postulate that La’+ mimics the effect of this altered subunit composition in the WT channel. The identification of the primary defect in the trp mutant at the level of the pore of a light-sensitive channel clearly raises the possibility that the channels are encoded, at least in part, by the trp gene. This proposal [52] is supported by the trp gene sequence [65]. Thus, as recognised by Phillips et al. [66], putative transmembrane regions of the rrp sequence show weak but significant homologies with known channel genes, the closest homology being with vertebrate voltage-gated Ca2+ channel asubunits (dihydropyridine receptor, DHPr). However, unlike the DHPr which contains 4 repeated transmembrane domains, each with 6 putative membrane spanning helices, the trp sequence represents only one such domain, and by analogy, (e.g. with K channels), it seems likely that the native channel is made up of multiple (probably 4) subunits (reviewed

in [67]). Phillips et al. [66] identified a second putative channel gene, trpl, also expressed in Drosophila photoreceptors and showing ca 40% overall identity with trp. Whether the native channels are homomultimers (e.g. 4 repeated trp subunits) or heteromultimers including subunits encoded by both trp and trpl or other, as yet unidentified, genes is unclear. Interestingly, both trp and trpl contain one or more calmodulin (GM) binding sites suggesting a role for Ca/CaM in channel activation or regulation. They also include an ankyrin (ANK) repeat motif which in other proteins is believed to represent a site for protein-protein interactions such as cytoskeletal attachment [68]. The trp sequence also contains an unusual proline-rich sequence near the C-terminal; a similar region in the bacterial protein TonB is thought to provide a mechanical linkage between proteins in the inner and outer membranes [69] leading to speculation that the TRP protein may span the short gap between the plasma and SMC membranes [70]. Further evidence that trp and trpf represent plasma membrane ion channels comes from recent heterologous expression studies in insect St9 cells [70-721 and Xenopus oocytes [73,74]. In St9 cells, both trp and trpf are reported to form functional channels when expressed alone, each with distinct ionic permeability profiles. Broadly consistent with in situ light-activated conductance data, the trp channels are highly Ca2+ permeable, whilst still passing large monovalent cation currents in the absence of Ca2+. By contrast, trpl generates a constitutively active non-selective cation permeable channel [7 1,721. Presently, there are insufficient data to compare the properties of the in situ and heterologously expressed channels more rigorously. Single channel

properties

There are no reports of single channel recordings of the Drosophila light-sensitive channels (or of the heterologously expressed putative channel genes), and indeed their presumed location at the base of the microvilli [75] would make them rather inaccessible to patch clamp analysis. However, following metabolic exhaustion, the light-sensitive channels become spontaneously active, resulting in a noisy inward current (rundown current RDC). Power

CELL

264

CALCRJM

al

light

+K-J/

b) rundown

_

-20 1 -60

F r c)

Zi

Fig. 5 Caz+ influx triggered

or by photorelease -20

mV.

mediates of caged

voltage

Ca*+ (c). Traces

is stimulated

control

response

is recorded

at -60

influx.

An initial

facilitation

(t)

following very

chronic

rapidly

by photorelease conditions

spontaneous

inactivated

(-)

of caged

the time constant

caged Ca**

Ca2+ dependent

by a hyperpolarizing

a photoreceptor

mV

$I----f

influx,

mV

with mV

after

first

of inactivation

shows

on left all to same scale. a step of light,

delivering

force

Subtracting

the two

(-)

(time

with

channels constant

a saturating

(-) is again extremely

spectra of the noise are fitted by simple Lorentzian functions consistent with random channel activity and suggesting that they are effectively uncoupled from the amplification steps of the transduction cascade [76]. The spectra are best fitted by the sum of two Lorentzians (effective mean open times ca 2 and 0.25 ms) indicative of two classes of channel or a single channel with two open states. With physiological divalent ion concentrations (1.5 mM Ca*+, 8 mM Mg*+) the estimated effective single channel conductance is ca 3 pS (at a resting potential of 60 mV). However, this may represent the average of two classes of channel. The estimated conductance is also very sensitive to the voltage-dependent Mg*+ block, and is likely to be considerably reduced as the cell depolarises [64]. By analogy with the vertebrate rod cGMP-gated channel [77], it has been suggested that the low effective conductance conferred by the Mg*+ block may be an adaptation to improve

(note

traces

(left)

constant

during

through

different

then stepped

a time

of the currents

for Ca*+ entry

(a) Right

and the voltage

by inactivation Ca2+ Influx

the time course

the driving

of the light-sensitive

by the enhanced Ca*+.

The figure

step to increase

throughout.

is followed activation

feedback.

to -60 reveals

scale):

starting

mV

to enhance

the current

‘rundown’:

stimulus

Ca2+ influx.

activated

to the cell Adapted

clamped from

inactivation at 60

mV

in Ca’+

(a,b)

channels

at a holding

an initially

ca 2 ms). (c) Ca*+-dependent

an increase

sensitive

of ca 25 ms. (b) A similar

metabolic

rapid (ca 2ms).

following the light

potential

of

A second

by the enhanced voltage

large inward

step

applied

current

is

can also be triggered throughout.

Under

these

[52,40].

the photoreceptor’s signal-to-noise ratio [64,76]. Thus, by allowing a greater number of channels to contribute to a given response, the relative contribution of stochastic channel noise is reduced (in proportion to the square root of the number of channels). Ca*‘-dependent feedback The channels, when activated spontaneously during rundown, are subject to a marked Ca’+-dependent inactivation, which can be conveniently triggered by applying a hyperpolarizing voltage step to increase the driving force for Ca*+ influx (Fig. 5). The inactivation is blocked both by removing extracellular Ca*+ or by buffering intracellular Ca*+ with BAPTA indicating that the inactivation is mediated by Ca*+ influx [78]. With normal extracellular Ca2+or the time constant of inactivation is in the range l-4 ms

PI-MEDIATED

al

PHOTOTRANSDUCTION:

‘21 IONS

kinetics of transduction, by contributing to both positive and negative feedback at several stages of the transduction cascade. Virtually all these effects are mediated by Ca*+ influx via the light sensitive channels and are absent or reduced in the trp mutant or in the absence of extracellular Ca2+ 14950,521. In this context, the high Ca*+ permeability of the trpdependent channels can be seen as having a profound functional significance. A full account of the regulatory sites is outside the scope of this review, but as well as the light-sensitive channels themselves, molecular targets of Ca*+ mediated feedback are believed to include PKC [48,50,79], the InsPs receptor [80], a kinase which is required for the CaM dependent phosphorylation of 49 kD arrestin [81,82], the InaD gene product [83] and rhodopsin phosphatase (r&C gene product, [81,84]). Other potential targets could include the ninaC-calmodulin complex [85]; phospholipase C [26, 861 and CaATPase (see also Fig 2, [5,67,87]).

bumps

flash

L

pA 100 ms 50

b) WT

Fig.

6

The

protein flash

inoC

kinase of light

response.

phenotype.

C (PKC, fails

to terminate

however,

the primary be traced

excitation to the failure

(b) Summation

sponse

in irtnC

very

bright

product)

normally.

process

from

Adapted

to prolonged

however from

WT (left).

quantum

of these bumps

in response

of eye-specific

the response

The rising

is unaffected

of individual

illumination

not shown).

In the absence

gene

is indistinguishable

(right).

([79]:

(a)

inaC

results dim

the response

265

& TRP GENE

to a

phase

of the

indicating

that

The defect

can

bumps

to terminate

in an enhanced illumination. decays

reWith

to baseline

[79].

(speeding up with hyperpolarization). Interestingly, when physiologically activated by weak light, Ca2+dependent inactivation of the photocurrent is much slower (ca 25 ms) and preceded by a pronounced transient positive facilitation (Fig. 5a). Following saturating illumination, inactivation, now triggered by photorelease of caged Ca2+, is again characteristically rapid (Fig. 5c, [40]). This may indicate that the channels are in different states under the various conditions, or that the time course of inactivation is determined by the local (endogenous) Ca*+ buffering conditions which may possibly be swamped during spontaneous activation or bright light. More generally, Ca*+ plays a crucial role in mediating adaptation and determining the gain and

Mechanism of excitation Despite advances in the characterization of the lightsensitive channels, their mechanism of excitation remains obscure and complicated by the suggestion that there may be at least two classes of channel not necessarily excited by the same mechanism or messenger. In Limulus, some authors have suggested that there may be additional parallel pathways of excitation, diverging at the level of the G-protein [88,89]. In Drosophila, however, any divergence would appear to be downstream from PLC, because of the absolute block of transduction in null mutants of the norpA gene. PLC generates two intracellular messengers, InsP3 and diacylglycerol (DAG). The latter is presumably involved in activation of PKC. However, in the ‘inactivation but no afterpotential’ (inaC) mutant which lacks eye-specific PKC [48] excitation appears unaffected, although there are severe defects in response termination (Fig. 6, [50,79]). Although alternative actions of DAG, e.g. as a precursor of arachidonic acid (AA), cannot be completely excluded, it is generally assumed that only InsPs is required for excitation; however, this has not been rigorously demonstrated. Since the major known action

266

of InsPj is to release Ca*+ from intracellular stores, it has been hypothesised that the released Ca*+ is a messenger of excitation, either directly gating the light sensitive channels, or acting via an intermediate [67,90,91]. If a rise in cytosolic Ca*+ is the messenger of excitation, a number of criteria need be fulfilled: (i) light should release sufficient Ca”+ rapidly enough to account for excitation; (ii) raising cytosolic Ca*+ should mimic excitation; and (iii) preventing a rise in Ca*+ (e.g. with Ca*+ buffers) should block excitation. Currently, there is some dispute in the literature as to whether these criteria are met in any invertebrate photoreceptor, and, in addition, there may be qualitative differences between Drosophila, in which none of these criteria have been satisfactorily fulfilled, and Limulus in which there is some evidence to support all three. Ca*+ release and stores In Limulus photoreceptors, there is clear evidence that both light and InsP3 release Ca*+ from internal stores, raising cytosolic Ca*+ from submicromolar levels to at least 50 pM [92-941. The stores correspond to an extensive system of ‘submicrovillar cisternae’ (SMC) which represent a specialized compartment of the smooth endoplasmic reticulum [9,93,95]. Baumann and Walz [IO] have also convincingly demonstrated that the SMC in an insect photoreceptor (honey bee drone) release Ca*+ upon InsPy application in a permeabilised slice preparation. Using fluorescent Ca*+ indicators, Walz et al. [96] also obtained evidence for a light-induced release of Ca*+ from internal stores in the bee; although this could not be unequivocally distinguished from Ca*+ influx via the light-sensitive channels. In fly photoreceptors, SMC can be detected in EM sections in both Calliphora [97] and Drosophila (e.g. [98]); however, particularly in Drosophila, these are much smaller than in the bee or Limulus. Evidence that these represent Ca*+ stores comes from accumulation of Ca oxalate precipitates in EM sections from Calliphoru [97], but InsP3-sensitive release of Ca2+ from these stores has not been tested in flies. Several unsuccessful attempts have been made to detect light-induced release of Ca*+ in Drosophila using fluorescent Ca*+ indicators. Although a massive light-induced rise in Ca*+ can be detected,

CELL

CALCIUM

this signal is abolished in the absence of extracellular Ca*+ indicating that the measured rise could be entirely attributed to Ca*+ influx (Fig. 3, [39,51]). This failure to detect release may, however, represent a signal detection problem due to the small size of the putative stores, combined with well-buffered and localized release. Despite the lack of direct evidence for light- or InsPg-sensitive Ca*+ stores in Drosophila or other flies, indirect evidence for the involvement of Ca*+ stores comes from a consideration of the trp and inaC phenotypes. The original phenotype of the trp mutation was described as the complete collapse of the response during prolonged illumination (Fig. 4), with sensitivity recovering only slowly in the dark. Detailed investigations indicated that the response decay was due to the exhaustion of excitation rather than, for example, excessive adaptation [99]. These studies led to the proposal the trp gene product (TRP) is a Ca*+ transporter which refills the InsP3sensitive Ca*+ stores during light. Crucially this model assumes that Ca*+ release from the stores is required for excitation of the channels remaining in the mutant, and when TRP is absent (or inhibited by La’+), the Ca*+ stores are readily depleted during light causing the collapse of the light response. The slow recovery of the light response (-1 min) in the dark would then be explained by a less efficient refilling process [63]. Bright illumination also leads to the collapse of the light response in null mutants of the inaC gene [79] which encodes an eye-specific PKC [48]. Normally very bright lights are required to inactivate the response in inaC, however, in the absence of extracellular Ca*+, much weaker illumination rapidly causes response decay and loss of sensitivity which can be rapidly restored by exposure to extracellular Ca*+ [79]. Paradoxically, in normal Cae, sensitivity in in& is actually enhanced at low light levels. This increase in sensitivity can be traced to the abnormally slow termination of individual quantum bumps which consequently sum to give a larger, but more slowly inactivating macroscopic response (Fig. 6). Furthermore, as light levels are increased, the high gain is initially maintained and, although the light-induced increase in cytosolic Ca2+ is actually larger than in WT [53], the main manifestations of light adaptation, including the shift in V/log I curve,

PI-MEDIATEDPHOTOTRANSDUCTION:

CaIONS&TRP

GENE

are missing [79]. This suggests that PKC is required for the normal coupling between the increase in Ca2+ and light adaptation. These apparently contradictory manifestations of the in& phenotype can be reconciled on the assumption that the quantum bump corresponds to a process of quanta] release from Ca*+ stores, and that the defect in innC is due to a failure for the release to be effectively terminated. Consequently, following bright illumination, or in the absence of extracellular Ca*+, the response would collapse due to emptying of the stores, whilst at weak to moderate intensities, responses are facilitated due to the longer bumps. Since the primary defect in iriaC appears to be on the waveform of the quantum bump, the site of action of PKC is likely to be downstream from PLC, and it has been suggested that it is required for controlling Ca2+ sequestration or the negative feedback of Ca*+ on Ca2+ release [4,5.5 1,791. A trp-like decay of the light response can even be obtained in WT by prolonged (much longer relative to in&) exposure of the cell to Ca2+ free Ringer, a procedure which is likely to cause depletion of the Ca*+ stores (Fig. 4, [52]). A similar decay in WT is also observed during a critical developmental time window (ca 80-U% pupation) even in the presence of Cao and in spite of the expression of functional TRP protein [loo]. At this stage, it was also found that the photoreceptors were entirely unresponsive to light unless first provided with Ca2+ via the whole-cell recording patch pipette. This observation might be explained by assuming initially empty Ca*+ stores and a limited capacity of TRP in the refilling process at the pupa stage, despite the high Ca’+ permeability of the light-activated channels. Although Ca’+ permeability is reduced in trp, it is not abolished [52], and Ca-indicator dyes and Caselective electrode measurements (Fig. 3, [39,53]) indicate that there is a significant residual light-induced Ca”’ influx in the trp mutant which can reach some tens of pM following intense illumination. Furthermore, raising extracellular Ca2+ does not prevent the collapse of the response (Minke and Hardie unpublished observations). This suggests that the response decay in the trp mutant is not simply due to the block of Ca*+ entry into the cytosol per se. To reconcile the original hypothesis [63] with these

267

findings, one needs to assume that rapid and efficient refilling of the stores can only occur via the TRP influx pathway, as might be expected, for example, if the TRP-dependent refilling process requires interactions between TRP and proteins of the Ca2+ stores @MC). In summary, substantial evidence demonstrates the existence of light- (Limulus) and InsP3-sensitive (bee and Limulus) Ca*+ stores in other arthropod photoreceptors. Whilst there is no direct evidence for such stores in Drosophila, it seems unlikely that Drosophila has evolved a profoundly different mechanism of excitation from other insects, and the collapse of the response under various conditions where Ca2+ appears to be limiting (trp, inaC, Las+ block, Ca2+ free Ringer and critical pupal stage; Fig. 3) is most economically explained in terms of exhaustion of Ca*+ stores. Is Ca sufficient for excitation? This question has been addressed in Drosophila by photorelease of caged Ca*+ using DM-nitrophen loaded into photoreceptors via whole-cell recording pipettes [40]. To avoid a response to the photolytic flash itself, ‘blind’ mutants lacking rhodopsin or PLC were used. A protocol which raised Ca*+ from < 1 pM to ca 20-50 l.tM in ca 1 ms failed to activate any light-sensitive channels. This negative result was strengthened by two positive controls. Firstly, the caged Ca*+ reliably activated a high capacity electrogenic Na/Ca exchange current. Secondly, in WT photoreceptors, when the caged Ca2+ was released during the rising phase of the response to light, it profoundly modulated the light sensitive conductance with a sub-millisecond latency (Figs 5 & 7). Interestingly, the polarity of this modulation depended critically on the timing of release: when released during the rising phase of the response, the caged Ca2+ greatly facilitated the response to light, but during the later ‘plateau’ phases, it induced a rapid inactivation. These results, therefore, suggest that Ca*+ is not a sufficient messenger of excitation in Drosophila, but confirm earlier studies showing dual positive and negative feedback roles for Ca2+. It should be pointed out, that the situation in Linzulus ventral photoreceptors may be different, since injections of Ca2+ do indeed activate a current

CELL CALCIUM

268

with similar properties to the light-activated current in this species [90,91]. Is Ca2+ required for excitation? Results of the converse experiment, namely attempting to block excitation with Ca2+ buffers, are equivocal. In normal (1.5 mM) Ca2+ Ringer, loading cells with BAPTA greatly reduced sensitivity and slowed down the response kinetics. The latter effect is to be expected when the Ca2+-mediated negative feedback responsible for rapid response termination is blocked [91]. However, when experiments were performed in Ca’+-free Ringer, to eliminate Ca2+ influx, the concentration of BAPTA required to attenuate sensitivity was significantly higher than in the presence of extracellular Ca2+. Furthermore, the BAPTA no longer slowed down the response kinetics (which were in fact slightly accelerated) suggesting that if any Ca2+ is released under these conditions it is insufficient to generate negative feedback [ 1011. These results are in marked contrast to the measured changes in cytosolic Ca2+ during the response to light - i.e a massive light-induced Ca2+ influx in the presence of extracellular Ca2+, and no detectable release in Ca2+ free Ringer (Fig. 3, [39,51]). Assuming that the effects of BAPTA in Ca2+-free Ringer’s are attributable to chelation of released Ca2+, the high concentrations required imply very short diffusion times, i.e. a very precise co-localization of Ca2+ release sites and their molecular targets. However, the possibility that some of the effects of BAPTA are attributable to alternative pharmacological sites of action should not be excluded. In particular, it may be relevant that BAPTA has been reported to act as a competitive antagonist of the InsPj receptor [ 1021. With respect to the criteria originally addressed therefore, in Drosophila: (i) there is no direct evidence for light-induced release of Ca2+ from internal stores although excitation fails under conditions when stores might be expected to be depleted; (ii) raising cytosolic Ca*+ fails to activate the light-sensitive channels although it profoundly modulates their activity after they have been activated physiologically; and (iii) as just mentioned, the results of preventing Ca2+ release are equivocal. In view of this limited support, although the hypothesis that cy-

Na+

b)

l-‘,“rn:”

WT

light

--I

contra’ ------xc I

500 pA 5 ms

L

Fig. 7 Caged Ca*+ fails to activatethe light-activatedconduct-

ance.(a) In the ninaE mutant which lacksrhodopsin,photorelease of caged Ca*+ (arrow) induces a small rapid inward current in the presence of normal Ringer (Na+). However, when Na+ is replaced by Li’ this response is abolished suggesting it represents activation of electrogenic Na/Ca exchange. (b) Positive control indicating that the caged Ca*+ has access to the light-sensitive channels: in a WT photoreceptor, caged Ca*+ released during the rising phase of the response to saturating illumination induces pronounced facilitation. In a control cell containing no caged Ca*+ the same flash used to release the caged Ca*+ has no effect beyond a transient biphasic response which is the ‘early receptor current’ (i.e. the charge movement in the membrane resulting from the conformational change of metarhodopsin to rhodopsin conversions). When released during the plateau phase of the response, caged Ca*+ also causes pronounced inactivation of the light sensitive channels (see Fig 5~). Adapted from [40].

tosolic Ca*+ is a messenger of excitation should not be abandoned, it would seem advisable to explore alternative hypotheses.

PI-MEDIATEDPHOTOTRANSDUCTION:Ca Alternative

IONS&TRP

GENE

hypotheses

CGMP Despite extensive evidence supporting the PI cascade in invertebrate phototransduction, in Lirnulus there is also evidence suggesting that the light-sensitive channels may be gated by cGMP. In particular, in.jection of cGMP or analogues can mimic excitation and excised patches containing light-sensitive channels can be activated by cGMP [103,104], whilst pharmacological agents affecting cyclic nucleotide metabolism can influence the response to light [88]. A role for cGMP has also recently been suggested in Drosophila, namely a cyclic nucleotide gated (CNG) channel has been cloned from a Drosophila cDNA library and has been reported to be expressed in eye tissue [lO5]. When heterologously expressed in Xertopus oocytes, its properties show some intriguing similarities to the native light-activated conductance. In particular, it has a similar high Ca*+ permeability and is subject to a voltagedependent block by divalent ions [ 1051. However, the suggestion that the light-sensitive channels correspond to the Drosophila CNG channel is difficult to reconcile with the effect of the trp mutation which greatly reduces both the Ca*+ permeability [52] and the Mg*+ block [64], so that the residual light-sensitive conductance in the trp mutant no longer bears any close resemblance to the Drosophila CNG channel. In principle though, the possibility that the trp gene product is required for the activation of a CNG channel should not be excluded. Capncitative Ca2+ entry The finding that the light sensitive channels in Drosophila are particularly permeable to Ca*+ suggests an analogy with a phenomenon reported in a variety of non-excitable cell types in vertebrates. Namely it has been known for many years that activation of the PI cascade is almost invariably associated with the opening of Ca*+ channels in the plasma membrane and that the resulting Ca*+ influx is required for refilling the stores and a maintained physiological response. As in invertebrate phototransduction, the mechanism of activation of this influx pathway

269

is unresolved and the subject of some controversy, but the most influential model is that the relevant signal is not a rise in cytosolic Ca*+, but the depletion of Ca*+ from the InsPJ-sensitive stores [55]. The evidence for this so-called ‘capacitative Ca*+ entry’ model comes from numerous experiments in which manipulations designed to deplete the Ca*+ stores result in activation of a Ca*+ influx pathway. These manipulations include application of Ca*+ ionophores (e.g. ionomycin) and microsomal Ca-ATPase inhibitors (e.g. thapsigargin) which block the Ca*+ pumps required for uptake into the store lumen. Although application of thapsigargin to Drosophila photoreceptors fails to activate the light-sensitive current ([51], Hardie unpublished observations, Cook and Minke unpublished observations), two lines of evidence now suggest that a capacitative Ca*+ entry-like mechanism may underlie the activation of at least a component of the light-sensitive current in Drosophila. Firstly, recent experiments have shown that ionomycin can activate the lightsensitive channels in the absence of extracellular Ca*+ and in the presence of high concentrations of internal BAPTA buffering [ 1061. Under these conditions internal stores should be depleted, without any change in cytosolic Ca*“. Secondly, although thapsigargin appears ineffective in Drosophila photoreceptors, recent experiments using trp heterologously expressed in an insect cell line (Sf9 cells [70]) and oocytes [73,74], show that a trp-dependent conductance can indeed be reliably activated by thapsigargin. Possibly the failure of thapsigargin to activate the channels in situ may indicate that the SMC have a specialised Ca*+ sequestering mechanism which is insensitive to thapsigargin, or that there is very little leak of Ca*+ from the SMC. Even if excitation is indeed an example of capacitative Ca*+ entry, this still does not provide the solution to the mechanism of invertebrate phototransduction. One is instead now faced with the same problem that has been facing the Ca*+ signalling community for years: how is the signal (presumed to be depletion of store Ca*+) transmitted from the store lumen to the plasma membrane? Currently there are two general classes of mechanism to explain this: one proposes a further diffusible messenger, so-called calcium influx factor or CIF

270

Fig.

CELL

8

influx

Cartoon channel

showing (TRP

by conformational tional’ Ca’+. leading

capacitative

changes

is coupled in the InsP3

Ca2+ entry

A Ca2+ binding to channel

the conformational

protein)

view,

receptor

gating

site on the luminal

eating.

However.

coupling

to the SMC

model membrane

which

would

it is also possible

by a direct

are transmitted

occur

terminus

of capacitative

as shown

of the InsPs

with

to the channel

in steps receptor

that InsPs alone might

[ 107.108]. Another, more radical hypothesis is that the Ca2+ entry channel may be linked to, and gated by, a protein-protein interaction with the InsP3 receptor (IP3R) and that Ca*+ binding sites - on the luminal terminus of the IP3R or a related luminal protein - can act as sensors for luminal Ca2+ (Fig. 8, [3,54,109]). This so-called conformational coupling model also allows for regulation of the influx pathway via modulation of various sites on the InsP3 receptor and channel. In this respect, it should be emphasised that whilst store depletion is usually an effective method for activating the Ca*+ influx pathway experimentally, there is in fact no direct evidence that this is actually the physiological signal for excitation of this pathway. For example, it is possible that the influx pathway may be triggered by

Ca2+ entry linkage 1-3, senses

[54,109]. the InsPs

According receptor

via a protein-protein

i.e. InsPs

causes

the reduction

be sufficient

to trigger

Ca*+ triggering influx

to this (IPsR).

model,

Gating

interaction. retease,

thereby

CALCIUM

In the ‘convenreducing

the conformational without

the Ca2+

is controlled

Ca2+ release

store change [4].

InsP3 alone without a requirement for release (Fig. 8(4), see also [109]) whilst luminal Ca*+, along with other modulators of the IP3R - most importantly, cytosolic Ca*+ [ 110,ll l] - may simply modulate the sensitivity.

CONCLUSION The proposed homology between invertebrate phototransduction and PI-regulated Ca*+ entry not only provides new ideas for understanding the longstanding enigma of invertebrate phototransduction, but, in addition, the unique molecular genetic potential of Drosophila may be expected to play an important role in unravelling the poorly understood

PI-MEDIATED

PHOTOTRANSDUCTION:

Ca IONS

& TRP GENE

phenomenon of capacitative Ca*+ entry. In particular, it should be noted that the molecular identity of the Ca*+ influx channels in vertebrates has yet to be revealed, and it is consequently possible that trp, as well as representing one component of the light sensitive current, may represent the prototypic member of a family of channels generally responsible for the widespread phenomenon of PI-regulated Ca*+ influx. In this respect, it is interesting to note that fragments of vertebrate trp homologues have recently been identified in mouse brain and Xempus oocyte cDNA libraries [73].

Acknowledgements We wish to thanks Dr M Berridge, SB Laughlin and Z Selinger for providing helpful comments on the manuscript. The authors’ researchis supported by National Institutes of Health (EY03529) and US Israel Binational Science Foundation (BM); The Royal Society, BBSRC and The Wellcome Trust (RCH).

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Neurosci.. 9. 87-l 19. 3. Berridge MJ. (1993) Inositol trisphosphate and calcium signailing. Nature. 361. 31 S-32.5. 4. Selinger Z. Doza YN. Minke B. (1993) Mechanisms and genetics of photoreceptors desensitization in Droso~hiln flies. Biochim. Biophys. Acta. 1179, 283-299. 5. Ranganathan R. Malicki DM. Zuker CS. (1995) Signal transduction in Drosophila photoreceptors. Annu. Rev. Neurosci.. 18. 283-317. 6. Clapham DE. (1995) Calcium signaling. Cell. 80, 259-268. 7. Lee Y-J. Shah S. Suzuki E. Zars T. O’Day PM. Hyde DR. ( 1994) The Drosophila &~y gene encodes a GAlpha protein that mediates phototransduction. Neuron, 13. 1143-I 157. 8. Schneuwly S. Burg MC. Lending C. Perdew MH. Pak WL. ( I99 I ) Properties of photoreceptor-specific phospholipase C encoded by rhe norpA gene of Drosophila me/nnog:cwer. J. Biol. Chem.. 266,24314-24319. 9. Fenp JJ. Carson JH. Morgan F. Walz B. Fein A. (1994) Three-dimensional organization of endoplasmic reticulum in the ventral photoreceptors of Linmlus. J. Comp. Neurol., 341. 172-183. IO. Baumann 0. Walz B. (1989) Calcium and inositol polyphosphate-sensitivity of the calcium-sequestering endoplasmic reticulum in the photoreceptor cells of the honeybee drone. J. Comp. Physiol., 165, 627-636.

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47.

48.

49.

50.

5 I.

52.

53.

54.

55. 56.

57. 58.

59. 60.

6 I.

62.

63.

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& Accepted

: I4 August

1995