purinergic signalling – a possible mechanism for kcnq1 channel response to cell volume challenges

Purinergic signalling – a possible mechanism for KCNQ1channel response to cell volume challenges

S. Hammami,1 N. J. Willumsen,1 A.-K. Meinild,1 D. A. Klaerke2 and I. Novak1

1 Department of Biology, University of Copenhagen, Copenhagen, Denmark

2 Department of Physiology and Biochemistry, IBHV, University of Copenhagen, Copenhagen, Denmark

Received 12 February 2012,

revision requested 11 March

2012,

revision received 9 May 2012,

accepted 28 May 2012

Correspondence: I. Novak,

Department of Biology, University

of Copenhagen, The August

Krogh Building, Universitetsparken

13, 2100-Copenhagen Ø,

Denmark.

E-mail: [email protected]

Abstract

Aim: A number of K+ channels are regulated by small, fast changes in cell

volume. The mechanisms underlying cell volume sensitivity are not known,

but one possible mechanism could be purinergic signalling. Volume acti-

vated ATP release could trigger signalling pathways that subsequently lead

to ion channel stimulation and cell volume back-regulation. Our aim was

to investigate whether volume sensitivity of the voltage-gated K+ channel,

KCNQ1, is dependent on ATP release and regulation by purinergic signal-

ling.Methods: We used Xenopus oocytes heterologously expressing human

KCNQ1, KCNE1, water channels (AQP1) and P2Y2 receptors. ATP

release was monitored by a luciferin–luciferase assay and ion channel con-

ductance was recorded by two-electrode voltage clamp.Results: The luminescence assay showed that oocytes released ATP in

response to mechanical, hypoosmotic stimuli and hyperosmotic stimuli.

Basal ATP release was approx. three times higher in the KCNQ1 + AQP1

and KCNQ1 injected oocytes compared to the non-injected ones. Exoge-

nously added ATP (0.1 mM) did not have any substantial effect on volu-

me-induced KCNQ1 currents. Nevertheless, apyrase decreased all currents

by about 50%. Suramin inhibited about 23% of the KCNQ1 volume sen-

sitivity. Expression of P2Y2 receptors stimulated endogenous Cl� chan-

nels, but it also led to 68% inhibition of the KCNQ1 currents. Adenosine

(0.1 mM) also inhibited the KCNQ1 currents by about 56%.Conclusion: Xenopus oocytes release ATP in response to mechanical sti-

muli and cell volume changes. Purinergic P2 and P1 receptors confer some

of the KCNQ1 channel volume sensitivity, although endogenous adenosine

receptors and expressed P2Y2 receptors do so in the negative direction.

Keywords adenosine, AQP1, ATP release, CaCC, P2 receptors, Xenopus

oocytes.

Adenosine triphosphate (ATP) is a highly hydrophilic

molecule responsible for energy storage inside the

cells. In the last decades, many studies have reported

a continuous basal release of ATP into the extracellu-

lar medium, where it can function as an autocrine/

paracrine signal molecule. Extracellular ATP interacts

directly with purinergic P2 receptors localized on cell

membranes, and after ATP degradation to adenosine

it interacts with P1 receptors. ATP modulates many

cellular functions, such as regulation of tissue blood

flow, growth, neuronal activity, epithelial transport

and response to pathogens (Corriden & Insel 2010,

Novak 2011). A number of cell types, both secretory

and non-secretory, release ATP upon activation by

© 2012 The AuthorsActa Physiologica © 2012 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2012.02460.x 1

Acta Physiol 2012

neuronal and hormonal agonists (Novak 2003, 2011,

Abbracchio et al. 2009, Corriden & Insel 2010), as

well as when exposed to mechanical stress and

changes in cell volume. Some studies also indicate an

important role of ATP in cell volume regulation (for

reviews, see Franco et al. 2008, Novak 2011).

It is well known that a number of K+ channels are

activated by small, fast changes in cell volume and

eventually contribute to cell K+ loss and volume back-

regulation (Grunnet et al. 2003, Calloe et al. 2007,

Hoffmann et al. 2009). Particularly, the voltage-gated

potassium channel KCNQ1 (also known as Kv7.1 or

KvLQT1) that assembles with KCNE1 to form cardiac

IsK has proven very sensitive to changes in cell vol-

ume (Grunnet et al. 2003) and is thought to have an

important physiological role in cell volume regulation

in cardiomyocytes (Calloe et al. 2007), mammary epi-

thelial tissues (van Tol et al. 2007), K+ secretory cells

in the inner ear (Wangemann et al. 1995) and liver

cells (Lan et al. 2006). A number of hypotheses have

been proposed on how the channel activity is modu-

lated during cell volume changes. One possibility is

that cell volume sensitivity is mediated by a membrane

stretch. We have recently provided evidence against

this hypothesis by demonstrating that cell membrane

stretch and cell volume change are two independent

mechanisms that can regulate the big conductance K+

channels (BK) and KCNQ1 channels respectively

(Hammami et al. 2009).

Another possible mechanism is that volume-activa-

tion of ion channels is mediated by an autocrine/para-

crine mechanism in which the volume-induced ATP

release activates signalling pathways that subsequently

lead to ion channel stimulation, as previously shown

for several types of ion channels (Hafting et al. 2006,

Franco et al. 2008, Corriden & Insel 2010, Novak

2011), including volume activated Cl� channels

(Wang et al. 1996, Roman et al. 1999, Perez-Samartin

et al. 2000, Darby et al. 2003) and K+ channels (Hoff-

mann et al. 2009).

In our laboratory, we use the Xenopus oocytes

expression system, which is particularly suited to

study ion channel activity during small and fast

changes in cell volume. This requires co-expression of

potassium channels with AQP1, as native oocytes are

devoid of water channels. Grunnet et al. (2002) have

shown that in non-AQP1 expressing oocytes, decreas-

ing or increasing the osmolarity of the extracellular

medium by 50 mOsm changed the cell volume by

<0.2%. In contrast, oocytes expressing AQP1

responded immediately to hypoosmotic and hyperos-

motic stimuli with significant changes in volume by

approximately 5% (Grunnet et al. 2002).

The aim of this study was to investigate whether

ATP release and components of purinergic signalling

are involved in volume-mediated response of KCNQ1

channels. For this purpose, KCNQ1 channels, the reg-

ulatory subunit KCNE1, AQP1 and P2Y2 receptors,

were expressed in Xenopus oocytes and membrane

currents as well as ATP release were monitored.

Materials and methods

Expression in Xenopus laevis oocytes

cDNAs coding for Aquaporin 1 (AQP1) and KCNQ1

were subcloned into expression vectors and expressed

in Xenopus laevis oocytes. The oocytes were prepared

as described earlier (Hammami et al. 2009) or pur-

chased from Ecocyte Bioscience (Castrop-Rauxel, Ger-

many). cRNA was prepared by in vitro transcription

(T3 and T7 mMessage machine kit from Ambion by

Life technologies, Carlsbad, CA, USA) from DNA tem-

plates (coding for AQP1 and KCNQ1) linearized with

Pst1 and XbaI (New England Biolabs, Ipswich, MA,

USA) for AQP1 and KCNQ1 respectively. cRNA was

extracted by MegaClear kit (Ambion). One microgram

per microlitre of cRNA was injected in oocytes, which

were then kept in a Xenopus physiological solution,

also called Kulori solution (in mM: 90 NaCl, 1 KCl, 1

MgCl2, 1 CaCl2, 5 HEPES–Tris, pH 7.4) at 19 °C.

ATP release measurements

ATP released from individual defolliculated oocytes

(non-injected, KCNQ1 injected and KCNQ1 + AQP1

injected) was monitored 3 days after RNA injection,

when the expression of channels was highest, using a

luciferin–luciferase bioluminescence assay (FLAA;

Sigma-Aldrich, Saint Louis, MO, USA), similar to the

procedure described by Maroto & Hamill (2001) with

slight modifications. Individual oocytes were placed in

a 96-well plate containing 45 lL of Kulori solution

and 5 lL of the Sigma ATP assay reagent in each well

(1 mg mL�1 luciferin-luciferase mix). There was one

oocyte per well. As oocyte handling and transfer to the

well was expected to elicit an increase in ATP release

(see Results section), oocytes were left to rest in the

well for 1 h before ATP release was monitored using

Victor luminometer (Perkin Elmer, Waltham, MA,

USA). After this first blank measurement, 22.5 lL of a

50% Kulori solution (Kulori diluted with

water) + 2.5 lL luciferin–luciferase mix (LL-mix) or

22.5 lL Kulori solution (Kulori with 100 mM manni-

tol) + 2.5 lL LL-mix was added carefully by pipetting

to swell or shrink the oocyte respectively (Δ20–30mOsmol change). For control, 22.5 lL of Kulori solu-

tion + 2.5 lL LL-mix was added in order to subtract

any pipetting effect. The ATP release was measured

immediately hereafter. Mechanical stimuli were evoked

© 2012 The AuthorsActa Physiologica © 2012 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2012.02460.x2

Purinergic signalling and KCNQ1 channels · S Hammami et al. Acta Physiol 2012

by pipetting Kulori solution into the well. Paired mea-

surements were taken on oocytes before and after

mechanical or osmotic stimuli. For both standards and

oocyte experiments, the ATP induced luminescence

was measured over a 60 s sampling period. The back-

ground signal (a blank) was measured and subtracted

from samples. Standard curves were performed by

plotting the ATP concentrations (M) against the lumi-

nescence intensity (relative luminescence units). The

Sigma ATP calibration standards were used with the

different osmolarities. The luminescence was converted

into M of ATP concentration according to the standard

curves prepared on each day of experiments.

Electrophysiological measurements

All measurements were taken 3 days after RNA injec-

tion using a conventional two-electrode voltage-clamp

set-up at a temperature range between 19 and 22 °C.The measurements were done in medium that was iso-

tonic (65 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM

CaCl2, 50 mM mannitol, 5 mM Hepes, pH 7.4

(188 mOsmol kg�1), hypoosmotic (65 mM NaCl,

1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM Hepes,

pH 7.4 (138 mOsmol kg�1) or hyperosmotic (65 mM

NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 100 mM

mannitol, 5 mM Hepes, pH 7.4 (238 mOsmol kg�1).

The chemicals Apyrase, Suramin, PPADS (Pyridoxal

phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium

salt hydrate), Adenosine and ATP (Sigma-Aldrich) were

added to the different solutions in concentrations as

indicated. The recording pulse protocol was 8 s at

�80 mV and 4 s at +40 mV with a holding potential of

�80 mV. During the whole experiment, oocytes were

constantly perfused with the different solutions while

continuously subjected to the pulse protocol. Current

traces for figures were selected after around 80 s of per-

fusion with the new solution where the current had time

to stabilize. No changes in leakage currents were

observed during the different experimental conditions.

In ion channels expressing oocytes the signal-to-noise

ratio (currents measured in channel expressing oocytes

vs. native oocytes) was >70 (Furthermore, currents due

to expressed channels were much larger compared to

endogenous currents.). Ag/AgCl reference electrode was

used. Data acquisition and analysis was performed with

CLAMPEX 10 and CLAMPFIT 10 (Molecular devices, Sunny-

vale, CA, USA) software programs respectively. GRAPH-

PAD PRISM 4 (GraphPad software Inc., La jolla, CA,

USA) was used for preparing graphical displays.

Statistics

If nothing else mentioned, numerical data are

presented as means ± SEM with n observations in

different oocytes. In some series of experiments,

currents were normalized in relation to currents in

iso-osmotic solution. Comparisons are made by using

Student’s two-tailed, unpaired or paired t-test depend-

ing on the data.

Results

Basal ATP release at rest and during cell volume

challenges

To determine whether ATP release is stimulated during

cell volume challenges in Xenopus oocytes under vari-

ous osmotic conditions, the luciferin–luciferase biolu-

minescence assay was used. Initially, several control

experiments were carried out. Figure 1a shows stan-

dard curves of bioluminescence assay in Kulori control

solution and in hypoosmotic and hyperosmotic

AQP1 + K

CNQ1

2.0 × 10–8

100 000(a)

(b)

10 000

1000

100

10

1

0.1

0.01

1.5 × 10–8

1.0 × 10–8

5.0 × 10–9

0

10–13 10–12 10–11 10–10 10–9 10–8 10–7

ATP (M)

ATP

(M)

Figure 1 (a) Standard curves of the bioluminescence assay in

Kulori solution, hypoosmotic and hyperosmotic solution.

RLU denotes the relative luminescence units. (b) Basal ATP

release difference from non-injected oocytes (CTRL n = 116)

and injected oocytes (AQP1 n = 31, KCNQ1 n = 97 and

KCNQ1 + AQP1 n = 115) at resting condition. The data

represent mean ± SEM. Oocytes were from seven different

frogs. ***P < 0.001; NS, non-significant (unpaired t-test).


Acta Physiol 2012 S Hammami et al. · Purinergic signalling and KCNQ1 channels

solutions. The assay sensitivity was not reduced in the

different solutions. Immediately after handling and

transfer of oocytes to the wells we noticed ATP release

because of the mechanical perturbations, which abated

with time (not shown). Consequently, the oocytes were

allowed to rest before the experiments commenced.

Thus, basal ATP release was measured 1 h after oocytes

were transferred to a well containing Kulori solution

and luciferin–luciferease mix. Figure 1b shows the dif-

ference in basal ATP release at rest between non-

injected oocytes (CTRL) and oocytes injected with

AQP1, KCNQ1 or AQP1 + KCNQ1. AQP1-injected

oocytes had nearly the same basal ATP release as the

non-injected ocytes, but surprisingly, basal ATP release

of KCNQ1 injected oocytes was 3- to 4-fold higher.

We predicted that oocytes expressing AQP1, which

can rapidly undergo changes in cell volume (see Intro-

duction), would release ATP to the surrounding med-

ium at a higher rate within the first minutes compared

to the controls during osmotic challenges. To test this

hypothesis, ATP release was monitored before and after

pipetting hypo- or hyperosmotic solution to each well.

As pipetting itself may induce mechanical ATP release,

we first monitored ATP concentrations after simple

addition of Kulori solution to each well (Fig. 2a). In

non-injected oocytes pipetting had no significant effect

on ATP release, whereas it increased ATP release in

oocytes injected with KCNQ1 and AQP1 together and

with only KCNQ1. Figure 2b shows similar experi-

ments, now with addition of hypoosmotic solutions. In

non-injected oocytes, the hypoosmotic challenge

induced a very small release of ATP compared to oo-

cytes subjected to Kulori solution (3.8 and 3.2 nM

respectively). In oocytes expressing only KCNQ1, the

hypoosmotic challenge increased ATP release by about

32% (compare black and white bars for KCNQ1). In

AQP1-injected oocytes, which undergo cell volume

changes (Grunnet et al. 2002), we a priori expected a

larger ATP release when challenged with low osmolar-

ity. However, in the KCNQ1 + AQP1 injected oocytes,

a 36% release of ATP to the extracellular medium was

observed when oocytes were subjected to hypoosmotic

extracellular solution.

In another experiment (Fig. 2c), hyperosmotic expo-

sure, and hence expected cell shrinkage, also induced

ATP release in KCNQ1 + AQP1 and KCNQ1

expressing oocytes. Furthermore, a significant ATP

release was seen for the non-injected oocytes in

response to hyperosmotic challenge.

In summary, the data in Figure 2 show that native

oocytes release ATP in response to mechanical stress

and to alterations in extracellular tonicity. This ATP

release occurs regardless of the presence of AQP1 and

can therefore not be secondary to cell volume changes.

The question whether ATP release is coupled to the

volume sensitivity of KCNQ1 channels is addressed in

the following section.

Effect of added extracellular ATP on KCNQ1 currents

As already mentioned, KCNQ1 channels are very sig-

nificantly modulated by cell volume changes induced

4.0 × 10–8(a)

(b)

(c)

3.0 × 10–8

2.0 × 10–8

1.0 × 10–8

0

ATP

(M)

4.0 × 10–8

3.0 × 10–8

2.0 × 10–8

1.0 × 10–8

0

ATP

(M)

4.0 × 10–8

3.0 × 10–8

2.0 × 10–8

1.0 × 10–8

0

ATP

(M)

Figure 2 ATP release before and after exposing oocytes to

(a) mechanical stimuli (pipetting Kulori solution), and pipett-

ing of (b) hypoosmotic and (c) hyperosmotic solutions. Black

bars (Kulori) indicated control resting conditions in Kulori

solution. In (a), pipetting had a slight increasing effect on

ATP release in KCNQ1 and KCNQ1 + AQP1 injected

oocytes. In (b), hypoososmotic solution induced a higher

ATP release in the injected oocytes independent of whether

AQP1 was present or not. In (c), hyperosmotic solutions had

similar effect on ATP release in all cases. The data represent

the mean ± SEM (oocytes from seven different frogs) (Kulori

solution n = 35–40, Hypo n = 36–44 and Hyper n = 26–34).

NS, non-significant; *P < 0.05, **P < 0.01 and ***P < 0.001

(Paired t-test).



by Δ50 mOsmol change; cell swelling increases the

voltage-activated KCNQ1 currents, whereas cell

shrinkage decreases the currents (Grunnet et al.

2003). To investigate whether the response of

KCNQ1 conductance to predicted changes in cell vol-

ume involves the purinergic signalling pathway, we

added 100 lM ATP to the extracellular solution (iso-,

hypo- and hyperosmotic solutions) and currents were

recorded before and after the addition of ATP at the

end of +40 mV depolarizing potential (from a holding

potential of �80 mV). Figure 3 shows that 100 lMextracellular ATP had no effect on volume-induced

changes in the KCNQ1 current. Higher concentrations

of ATP were also tested with no effect detected (data

not shown). One explanation of the absence of exoge-

nous ATP effect could be that there is already a sub-

stantial ATP release, for example, the mere expression

of KCNQ1 channels in itself seems to increase ATP

release from the oocytes (cf. Fig. 1b).

Effect of apyrase on KCNQ1 currents

To further examine whether the response of the KCNQ1

currents to volume challenges could be mediated by

release of ATP, we added the ATP/ADP hydrolysing

enzyme, apyrase, at the different osmotic conditions

described earlier. Figure 4a,b shows that addition of

7 U mL�1 apyrase decreased the overall KCNQ1 current

levels in control, hypo- and hyper-osmotic conditions.

(a)

(b)

Figure 3 Effect of extracellular ATP on KCNQ1 current

response to cell volume challenges (co-expressed with AQP1).

(a) Original KCNQ1 + AQP1 current traces at 40 mV depo-

larizing potentials of 4 s duration (Vhold: �80 mV) during

osmotic challenges with and without the presence of 100 lMATP. (b) Columns show the changes of current at the end of

a depolarization period (+40 mV) during osmotic challenges

before (Control) and after addition of 100 lM ATP. The data

represent mean ± SEM n = 6 oocytes (NS: Non-significant

vs. control conditions, paired t-test).

Apyrase 7 U mL–1

Apyrase 7 U mL–1

(a)

(b)

(c)

Figure 4 Effect of application of extracellular apyrase

(7 U mL�1) on KCNQ1 current response to cell volume chal-

lenges. (a) Original KCNQ1 + AQP1 current traces at

40 mV depolarizing potential of 4 s duration during osmotic

challenges with and without the presence of apyrase. (b) Col-

umns show the changes of current at the end of a depolariza-

tion period (+40 mV) during osmotic challenges before

(control) and after addition of apyrase. The data represent

the mean ± SEM n = 5 oocytes (**P < 0.01, ***P < 0.001

vs. control conditions, paired t-test). (c) Normalized currents

(normalized to isoosmotic current (±Apyrase).



The currents also decreased further with higher concen-

trations of apyrase (data not shown). To exclude that the

mere presence of extracellular protein should affect the

KCNQ1 currents, a similar experiment was performed

with the addition of bovine serum albumin to the Kulori

solution at similar concentrations, but no effects were

observed (data not shown). When we normalized the

currents to the currents measured at isotonic conditions

(Fig. 4c), the relative percentage changes with hypo-

osmotic or hyperosmotic solutions were the same in

presence and absence of apyrase. Taken together, these

data show that the volume sensitivity of KCNQ1 was

unchanged in the presence of extracellular apyrase,

although the enzyme decreased the KCNQ1-currents in

all conditions, which may be due to the hydrolysing

effect of the enzyme on the basal ATP release close to the

membrane of KCNQ1-injected oocytes.

In the next series of experiments, we examined

effects of broad acting P2 receptor blockers, which

could affect P2 receptors possibly expressed endoge-

neously in Xenopus oocytes. A non-selective P2 recep-

tor antagonist, PPADS, was used at 0.1 mM

concentration, and results are shown in Figure 5a.

PPADS did not have any significant effect on the

KCNQ1 current during cell volume challenges. In

another series of experiments, we applied a non-selec-

tive purinergic receptor antagonist Suramin (Fig. 5b).

In these experiments, hypoosmotic extracellular med-

ium induced a lower current increase in oocytes trea-

ted with 0.1 mM Suramin compared to control

oocytes, namely, 69% compared to 121% increase

above control respectively (n = 14, P = 0.0001).

Application of 0.1 mM Suramin to hyperosmotic

extracellular medium induced a larger decrease in cur-

rent (56%) compared to hyperosmotic condition with-

out Suramin (43%).

Taken that Suramin did decrease, but not abolished

KCNQ1 response to cell volume challenges, indicates

that some endogenous P2 receptors may be involved

in volume-induced current changes – although to a

minor extent. Therefore, we considered P2Y2 recep-

tors and in a first set of experiments expressed the

P2Y2 receptor together with AQP1 to investigate

whether there is any interaction between purinergic

receptors and endogenous ion channels in Xenopus

oocytes exposed to cell volume challenges. Then in a

second set of experiments, oocytes co-expressed the

receptor P2Y2 and AQP1 together with KCNQ1 and

for both batches of oocytes, the same series of experi-

ments was performed as described earlier. Figure 6

shows that the expression of P2Y2 together with

AQP1 in Xenopus oocytes induces an upregulation of

an endogenous outwardly rectifying current (Fig. 6a).

When substituting extracellular Cl� with gluconate,

the current decreased and the reversal potential shifted

to more positive potentials (Fig. 6b). In addition,

when we substituted extracellular Ca2+ with Mg2+,

the current decreased (Fig. 6c). These results indicate

that expression of P2Y2 receptors upregulates the

endogenous Ca2+-activated Cl� channels in Xenopus

oocytes. In experiments where the P2Y2 and AQP1

expressing oocytes were subjected to different osmo-

larities, the endogenous Ca2+-activated Cl� currents

increased upon hyposomotic solutions and decreased

upon hyperosmotic conditions compared to oocytes

only expressing P2Y2 receptors (Fig. 6d,e). Notably

when coexpressing P2Y2 receptors with KCNQ1 and

Aquaporin, the endogenous currents were significantly

decreased in isoosmotic conditions (68% inhibition)

(3.21 ± 0.42 to 1.01 ± 0.17 lA, P = 0.0032) (cf.

Figs 4 and 7). The data in Figure 7 also depict the

effect of the P2Y2 receptor on KCNQ1 current

(a) (b)

NS

NS

Figure 5 Effect of extracellular PPADS and Suramin on KCNQ1 current response to cell volume challenges (coexpressed with

AQP1). Columns show the changes of current at the end of a depolarization period (+40 mV) during osmotic challenges before

(Control) and after addition of (a) 100 lM PPADS. The data represent mean ± SEM n = 5 oocytes. (b) 100 lM Suramin. The

data represent mean ± SEM n = 14 oocytes (**P < 0.05, ***P < 0.0001 vs. control conditions, paired t-test).



response to cell volume challenges. Importantly, in the

presence of the P2Y2 receptor, the KCNQ1 currents

only increased by 51% during cell swelling, whereas

in the absence of co-expressed P2Y2 receptor (but in

the presence of any endogenous P2 receptor) the

swelling induces increase in KCNQ1 current is 77%.

These observations indicate that P2Y2 receptors are in

fact inhibitory not only to KCNQ1 currents, but also

to the swelling-induced regulation.

The KCNQ1 channels are regulated by the KCNE1

subunit (Jespersen et al. 2005, Sanguinetti et al.

1996). Therefore, in one series of experiments, the

KCNE1 subunit was co-injected with KCNQ1 and

AQP1, and oocytes were exposed to changes in

osmolarity (Fig. 8a). The control currents and cell

swelling-induced currents were similar to those found

in oocytes expressing KCNQ1 alone. Suramin still had

a small inhibitory effect on hypoosmotically induced

current (Fig. 8b).

Lastly, we addressed the question as to whether the

ATP hydrolytic product, adenosine (0.1 mM), could be

involved in regulation of the KCNQ1 currents.

Figure 9 shows that adenosine suppressed the hy-

poosmotically mediated volume effects on KCNQ1

(a)

(b)

(c)

(d)

(e)

Figure 6 (a) Comparison between currents in AQP1 and AQP1 + P2Y2 expressing oocytes. Expression of P2Y2 in Xenopus

oocytes induces an upregulation of an endogenous outwardly rectifying channel current, which has a reversal potential at

approx. 0 mV. (b) I/V curve for P2Y2 expressing oocytes at different Cl� concentrations. When substituting Cl� with gluconate,

the reversal potential was shifted to more positive values. (c) Substitution of Ca2+ with Mg2+ decreased the current. This indi-

cates that the current is mediated by Ca2+-activated Cl� channels. Co-expression of the P2Y2 receptor and AQP1 in Xenopus

oocytes induces a Ca2+-activated Cl� current that is volume sensitive. (d) Effect of changes in extracellular osmolarity on oocytes

expressing P2Y2 and AQP1 (n = 21). Currents increased by 58% when exposed to hypoosomotic solution (D50 mOsmol kg�1).

(e) However, no significant effect upon changes in extracellular osmolarity was seen on oocytes expressing only P2Y2 (n = 7).

Paired t-test comparing with isoosmotic conditions (***P < 0.0001).



currents; in the absence of adenosine, an activation of

2–3 times of control was observed, whereas in the

presence of adenosine, the activation decreased to

about 1.5 times of control.

Discussion

In this study, we show that oocytes clearly release

ATP in response to both mechanical stimulation and

cell volume challenges. Purinergic signalling has effects

on KCNQ1-related currents, although these are com-

plex. In the following sections, we will discuss how

purinergic signalling is involved in KCNQ1 current

responses to cell volume changes.

Basal ATP release

Basal ATP release is thought to prime or tune signal-

ling pathways that can affect responses of cells to

other incoming stimuli (Corriden & Insel 2010,

Novak 2011). For non-injected oocytes, basal release

corresponded to an equivalent of 19 femtomoles of

ATP per oocyte in 50 lL of Kulori solution. In previ-

ous studies, a basal ATP release for non-injected

oocytes was also measured around 20 femtomoles

(Maroto & Hamill 2001). Note that these very small

concentrations were measured in relatively large bulk

volumes (50 lL). The ATP concentration may very

well, however, be larger in close proximity to the

oocyte surface, especially in between the microvilli

and the many foldings of the oocyte membrane.

Another possible explanation for the low ATP concen-

tration is the endogenous expression of the membrane

protein CD39, which hydrolyses ATP and ADP (Aleu

et al. 2003).

KCNQ1 and KCNQ1 + AQP1 injected oocytes had

a threefold higher basal ATP release than the oocytes

injected with AQP1 only (Fig. 1b). This would indi-

cate that the type of membrane protein expressed

might have different effects on the basal ATP release.

Heterologous expression of KCNQ1 channel may

have several consequences: (i) the oocyte is stressed

(a) (b)

Figure 7 Effect of P2Y2 injection on KCNQ1 current and its volume response. (a) Absolute values. (b) Currents normalized to

isoosmotic condition. The data represent mean ± SEM n = 4–5 oocytes. Paired t-test comparing with isoosmotic conditions

(*P < 0.05, **P < 0.01 and ***P < 0.001).

(a) (b)

Figure 8 (a) Effect of KCNE1 injection on KCNQ1 current and its volume response. The data represent mean ± SEM n = 5–6

oocytes. (b) Effect of 100 lM Suramin on KCNE1 + KCNQ1 + AQP1 expressing oocytes (**P < 0.01).



because of a high production rate of proteins and high

rate of exocytosis, which may include exocytotic ATP

release; (ii) KCNQ1 may be functionally interacting

and upregulating the insertion of ATP transporters to

the membrane; and/or (iii) KCNQ1 that would hyper-

polarize oocytes may be permeable to ATP. Several

mechanisms have been suggested regarding how ATP is

released in various cell systems. It can be released by

constitutive release of vesicles and through hormonal/

neuronal regulated exocytosis. Moreover, ATP release

can take place by a conductive ionic process through

membrane channels, such as mechanically gated ion

channels, hemichannels such as connexins and pannex-

ins, maxi anion channels, volume-regulated anion chan-

nels, CFTR and P2X7 receptors (for reviews, see

Corriden & Insel 2010, Novak 2011). It is unlikely,

however, that KCNQ1 constitutes an ATP conductive

channel, because ATP is negatively charged in physio-

logical solutions, while options 1 and 2 may be feasible

in our system. In fact, for Xenopus oocytes vesicular

and endogenous connexin Cx38 have been suggested

(Maroto & Hamill 2001, Bahima et al. 2006). Interest-

ingly, KCNQ1 expression that up-regulates ATP release

is overshadowing AQP1-dependent volume-induced

ATP release in Xenopus oocytes (Figs 1 and 2).

ATP release during cell volume challenges

ATP release was measured under iso-, hypo and hyper-

osmotic conditions for non-injected oocytes, KCNQ1 +AQP1- and KCNQ1-injected oocytes. Swelling-induced

ATP release is widely reported in many native and cul-

tured cells (Wang et al. 1996, van der Wijk et al. 1999,

Shinozuka et al. 2001, Boudreault & Grygorczyk

2004). However, previous studies where defolliculated,

non-injected Xenopus oocytes were tested in hypoos-

motic stress conditions (D140 mOsmol L�1) did not

detect any release of ATP (Maroto & Hamill 2001,

Aleu et al. 2003). In contrast, oocytes challenged with

hyperosmotic solution (D300 mOsmol L�1) released

ATP (Aleu et al. 2003). We also observed minimal

changes in ATP release in non-injected oocytes subjected

to hypoosmotic conditions (D20–30 mOsmol L�1), but

hyperosmotic solutions (D20–30 mOsmol L�1) dou-

bled the ATP release in our non-injected oocytes (cf.

Fig. 2b,c). During cell shrinkage, the released ATP in

Xenopus oocytes was shown to be coupled to the acti-

vation of inward currents sensitive to Gadolinium and

to inactivation of ecto-nucleotidase CD39, the latter

causing increase of the basally released ATP (Zhang &

Hamill 2000a, Aleu et al. 2003). It is possible that

different ATP release mechanisms operate under hypo-

osomotic and hyperosmotic changes and that different

purinergic receptors are activated and regulating differ-

ent ion channels.

In the injected oocytes (KCNQ1 and KCNQ1 +AQP1), we detected significant release of ATP above

control levels in both hypo- and hyperosmotic condi-

tions, as compared to control oocytes. How can we

explain the higher ATP release with KCNQ1 expres-

sion? One possibility is that when expressing an exoge-

nous channel, the exocytotic pathway is boosted. In

parallel, swelling the oocyte may intensify the response,

thereby contributing to a higher release of vesicles con-

taining both the channel proteins ready to be inserted to

the membrane and the ATP molecules ready to be

released to the outside. The question is whether this cor-

relates with expected cell volume changes. We would

have expected that the AQP1 + KCNQ1 expressing oo-

cytes, to be most volume sensitive and therefore we

would expect to see a higher ATP release. However,

AQP1 expression and associated volume changes does

not boost ATP release in the KCNQ1 injected oocytes.

This indicates that larger volume changes in the pres-

ence of AQP1 do not induce a higher release of ATP and

that the rate of release is independent on AQP1. Possi-

bly, we cannot detect small ATP changes in our bulk-

solution detection system and small osmotic challenges.

Alternatively, the AQP1-mediated change in oocyte vol-

ume is too modest [around 5%, (Grunnet et al. 2002)]

and/or oocyte CD39 activity is high, and therefore

effects are not as pronounced as seen in mammalian

cells exposed to hypoosmotic loads (Boudreault &

Grygorczyk 2004, Hoffmann et al. 2009).

Effect of exogenous ATP, apyrase and P2 receptor

antagonists on currents

In another series of experiments ATP, apyrase and P2

receptor antagonists were added to the extracellular

Figure 9 Effect of extracellular Adenosine (100 lM) onKCNQ1 current response to cell volume challenges (coex-

pressed with AQP1). Columns show the changes of current

at the end of a depolarization period (+40 mV) during osmo-

tic challenges before (Control) and after addition of 100 lMadenosine. The data represent mean ± SEM n = 5 oocytes

(*P < 0.05 compared to hypoosmotic control, paired t-test).



medium to investigate whether the response of

KCNQ1 to cell volume challenges involves ATP

release and a subsequent purinergic signalling path-

way. Apyrase decreased both basal- and volume-stim-

ulated currents. Either the effect was non-specific or

hydrolysis of basally released ATP/ADP close to

oocyte plasma membrane resulted in tuning down of

endogenous ion channels. Possibly adenosine accumu-

lation and adenosine receptors could have been

responsible for this effect.

Exogenous ATP at relatively high concentrations

had no extra effect on current responses (Fig. 3). This

could be because ATP concentrations were saturating

or because there were no P2 purinergic receptors

involved. The existence of endogenous purinergic

receptors in Xenopus oocytes is puzzling. Many stud-

ies based on electrophysiological experiments have

shown that follicular cell-enclosed oocytes are

endowed with purinergic and adenosine receptors

evoking endogenous Cl� and K+ current responses

(Lotan et al. 1986, King et al. 1996a,b, Arellano et al.

1996, 1998, 2009). Defolliculated oocytes are reputed

to express a few if any purinergic receptors, and this

was based on the observation that ATP failed to acti-

vate current responses. However, if such measure-

ments were taken a few hours after defolliculation of

oocytes, the receptors could be desensitized and/or

internalized. Possibly also source of animals and age -

may influence receptor expression. Nevertheless, there

are a few studies indicating that there are endoge-

nous adenosine and P2 receptors in the membrane

Xenopus oocytes (Kupitz & Atlas 1993, King et al.

1996a,b, Kobayashi et al. 2002). In our study, we

used oocytes 3–4 days after defolliculation and RNA

injection and tested for the presence of endogenous

P2 receptors and involvement of purinergic signalling

pathway in the KCNQ1 response to cell volume

challenges. Suramin, but not PPADS, had small but

significant effect on hypoosmotic response of

KCNQ1 and AQP1 expressing oocytes (the basal

currents were unchanged). This implies that some P2

receptors may be involved in this response. The iden-

tity of P2 receptor types expressed in the oocyte

membrane could be determined by the use of more

specific purinergic receptor blockers and molecular

identification.

Effect of P2Y2 and adenosine receptors on currents

We co-expressed the P2Y2 receptor, which is often

associated with regulation of Cl� and K+ channels

(Novak 2011). This receptor activated the endogenous

Xenopus oocyte Ca2+ activated Cl� channel (CaCC)

ascribed the TMEM16A chloride channel (Schroeder

et al. 2008). The P2Y2 receptor is linked to the Gaq/11

protein, which activates membrane bound phospholi-

pase C and eventually inositol triphosphate-mediated

release of Ca2+ that would stimulate CaCC. This

CaCC channel current in our experiments was up-

regulated during hyposomotic conditions and down-

regulated during hyperosmotic conditions. These

experiments again further support the notion that

release of ATP during cell swelling would act on puri-

nergic receptors expressed in Xenopus oocytes and

stimulate specific Cl� channels.

However, when the P2Y2 receptor was coexpressed

with KCNQ1, the receptor had an inhibitory effect on

volume sensitivity of the K+ channel. A similar nega-

tive modulation of other K+ channels, that is, BK (also

named KCNMA1), we have observed earlier in native

pancreatic cells and also in oocyte expression system

(Hede et al. 1999, 2005). Other K+ channels can be

positively regulated by purinergic signalling as demon-

strated by Kobayashi et al. (2002). In that study, it is

reported that ATP stimulated G-protein inwardly-rec-

tifying K+ channels in defolliculated Xenopus oocytes,

although adenosine was more potent, again indicating

that endogenous purinergic and adenosine receptors

are expressed.

Interestingly, also other studies show a coupling of

the KCNQ1 to purinergic signalling in native mamma-

lian tissues. The strial marginal cells and vestibular

dark cell of the inner ear of rodents express KCNQ1,

its auxiliary b-subunit KCNE1 and purinergic recep-

tors (P2Y4) at the apical membrane, and these recep-

tor/channel proteins seem to have an important

function in endolymph homeostasis and protection

from overstimulation (Lee & Marcus 2008, Housley

et al. 2009). KCNQ1/KCNE1 channel activity and

thus K+ secretion are down-regulated by several puri-

nergic pathways in rodents including the P2Y4 recep-

tors (Lee & Marcus 2008). In mouse cardiomyocytes

and oocyte expression system, P2 receptors up-regu-

late KCNQ1/KCNE1 channel activity (Honore et al.

1992). We have also co-expressed KCNQ1/KCNE1 in

oocytes, and hypoosmotic changes and suramin had

similar effects as in oocytes expressing only KCNQ1

(Fig. 7). Interestingly, the slowly activated delayed-

rectifier K+ current IsK (KCNQ1/KCNE1) in guinea-

pig ventricular myocytes is modulated by osmotic

changes (Missan et al. 2011). In light of our study

and those quoted above, it is interesting to speculate

to what extent the purinergic signalling is involved in

this modulation.

Above findings indicate that the signalling path-

ways from P2Y2 (or P2Y4) receptors that modulate

K+ channels might be different to those modulating

Cl� channels. It has been suggested that phophat-

idylinositol 4,5 biphosphate (PIP2) is an important

regulator of KCNQ channels and its depletion



reduces the channel activity (Loussouarn et al. 2003,

Zhang et al. 2003, Park et al. 2005, Lee & Marcus

2008). This may explain our findings that show a

decrease in KCNQ1 currents by the P2Y2 receptor

co-expression. In addition, it is suggested that P2Y2

receptors interact with multiple signalling pathways,

and this may also involve trans-stimulation of Gai

proteins (see Novak 2011). Interaction with Gai/o

protein signalling pathways could also explain our

data with adenosine. That is, adenosine down-regu-

lates the KCNQ1 currents. It is known that KCNQ1

currents are sensitive to cAMP levels (Kunzelmann

et al. 2001) and stimulation of endogenous A1 recep-

tors would, most often via stimulation of Gai pro-

teins and inhibition of adenyl cyclase, lead to

reduction of cAMP.

In addition to KCNQ1 channels, also KCNQ4

channels are volume sensitive and regulated by recep-

tors coupling to Gai/o proteins such as dopamine D2

receptors (Grunnet et al. 2002, Ljungstrom et al.

2003). Whether purinergic signalling will have similar

effects on this channel remains to be investigated. Cer-

tainly, in the central nervous system where KCNQ

channels are richly expressed, cell volume regulation

and ATP signalling would be of importance.

Taken together, we showed that oocytes release

ATP in response to mechanical and small hypo- and

hyperosmotic loads, which may be due to different

release mechanisms. Hypoosmotic challenge increases

KCNQ1 currents (not hyperosmotic ones), and there-

fore if ATP release is involved, purinergic receptors/

signalling leading to activation of these channels must

be specific. Exogenous ATP and apyrase have no

effect on KCNQ1 currents stimulated by hypoosmotic

load, perhaps because purinergic receptors are already

saturated with locally released ATP. Here one has to

mention that oocytes have very folded membranes

that could give rise to difficulties in accessing local

microenvironments (Zhang & Hamill 2000b), and

purinergic signalling it is certainly played out in such

microenvironments (Corriden & Insel 2010, Novak

2011). Suramin decreased hypoosmotically induced

KCNQ1 currents indicating that endogenous P2Y

receptors may have been involved, although it was not

the P2Y2 receptor, as the expression of this receptors

inhibited KCNQ1 currents. Similarly, also adenosine

and therefore adenosine receptors inhibited KCNQ1

currents. In contrast, Ca2+-activated Cl� channels

were activated by P2Y2 receptors, implicating multi-

ple signalling pathways for this receptor.

In conclusion, purinergic signalling (ATP and aden-

osine) is most likely involved in volume-regulated

KCNQ1 currents. We have revealed both up- and

down-regulating factors in hypoosmotically induced

KCNQ1 currents.

Conflict of interest

The authors declare that they have no conflict of

interest.

The authors thank Ms. Z. Rasmussen for expert technical

assistance. This work has been supported by the Danish Nat-

ural Science Research Council (10-085217, 09-060639 and

the Carlsberg Foundation (2006-01-0529) and (2007-01-

01656) and Medical Research Council (09-07328).

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