Effect of internal F- on activation of Cystic Fibrosis
Transmembrane Conductance (CFTR) regulator byforskolin
Autors: Claudia S. Haarmann, Sonja Stölzle, Cecilia Farre, Andrea
Brüggemann

Nanion Technologies GmbH, Erzgiessereistr. 4, 80335 München,
Germany

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Aim:
The aim of this study was to investigate the effect of internal F- on the activation of thecystic fibrosis transmembrane conductance regulator (CFTR) by forskolin in the wholecell patch clamp configuration. Experiments were conducted with Nanion’s fully
automated patch clamp device, the Patchliner©.

Introduction:
The

CysticFibrosis

Transmembrane

Conductance

Regulator(CFTR)

isa

phosphorylation regulated Cl- channel. In order for the channel to be active it needs to bephosphorylated. Subsequent binding of ATP to intracellular nucleotide binding sites
controls the opening and closing of the channel (Zhou et al., 2006).
Our standard internal solutions contain F-. Regulation of many ion channels does notseem to be affected by the presence of F- in the intracellular solution, however, that is nottrue for every ion channel. F-, for example, has been shown to activate adenylate cyclase(Eckstein et al., 1979). Forskolin – a commonly used CFTR activator – is known toactivate CFTR through activation of adenylate cyclase. This leads to phosphorylation(and hence activation) of CFTR by protein kinase A (PKA). It would be interesting to see
if F- can be used as a substitute for forskolin in its role as a CFTR activator.
In this report we summarize some preliminary measurements of CFTR currents in thewhole cell patch clamp configuration with the Patchliner© to obtain insight into theeffects of F- on CFTR currents.

Material and Methods:
Cells
Baby hamster kidney cells (BHK) expressing wt CFTR or untransfected BHK
Cell culture
Cells were cultured in DMEM-F12, 5 % FBS (+500 µM methotrexate for cells
expressing CFTR) in T-25 culture flasks. They were split every second to third day –when 50-100 % confluent. 10-20 % of the harvested cells were then plated in new flasks.
Standard Nanion protocols were used to harvest the cells.
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For measurements, cells were harvested when 60-90 % confluent (2-3 days after plating),again, according to Nanion’s standard protocol.

Whole cell patch clamp recordings
Whole cell patch clamp recordings were conducted according to Nanion’s standard
procedure with the 2- or 4- channel Patchliner©. All solutions used for patch clamp
measurements are listed in Table 1a & b. First, the sealing behavior and endogenous
currents of untransfected BHK cells were investigated under conditions where Na+- andK+-currents could be observed (standard(F-), standard external) and under conditions
where both Na+- and K+-currents are absent (Na/K -free internal and external).
CFTR currents were compared under conditions when internal F- (standard (F-)) waspresent and under conditions when the F- was replaced by Cl- (standard (Cl-)). Theseexperiments were conducted with standard external solution.
In all experiments cells were kept at a holding potential of -80 mV. Every 5 s a voltage
ramp (-100 mV to +100 mV) was applied and the current response to these voltage ramps
was recorded.
To compare currents under different conditions, currents at +95 mV were used. They
were determined as the mean current flowing in the 95 mV ± 0.5 mV interval. Data are
given as mean ± SEM. Data sets were considered as significantly different when astudent’s t-test revealed a p < 0.05.
Table 1a: Internal solutions
Standard (F-)
75 mM
10 mM
70 mM
2 mM
10 mM
10 mM
pH 7.2

KCl
NaCl
KF
MgCl2
EGTA
HEPES(KOH)

standard (Cl-)

Na+/K+ -free

145 mM
10 mM

KCl
NaCl

70 mM
70 mM

CsF
CsAsp

2 mM
10 mM
10 mM
pH 7.2

MgCl2
EGTA
HEPES
(CsOH)

5 mM
10 mM
10 mM
pH 7.4

MgCl2
CsEGTAHEPES(CsOH)All internal solutions additionally contained 5mM ATP.

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Table 1b: External solutions
Na+/K+-freestandard
160 mM
4.5 mM
1 mM
2 mM
5 mM
10 mM
pH 7.4NaCl
KCl
MgCl2
CaCl2
D-Glucose Monohydrate
HEPES(NaOH)

145 mM TEA Cl
10 mM

CaCl2

10 mM HEPES (Na+ salt)
pH 7.4 (CsOH)

Results
Characteristics of untransfected BHK cells
To test how the BHK cell line behaves on the Patchliner© and what currents the cells
endogenously express we conducted a set of control measurements with untransfected
BHK cells. Cells were subjected to the protocol that we used for the investigation of theCFTR currents: cells were automatically sealed and broken into by the predefined
Patchliner© protocol. Once a stable whole cell configuration was established voltage
ramps (-100 mV to +100 mV) were applied every 5 s and current responses recorded.
To observe what type of currents were present in the untransfected cells we used two setsof solutions: (1) Na/K - our standard solutions with external high Na+ and internal highK+, conditions under which Na+- and K+-currents can be observed; (2) TEA/Cs - solutions
in which the external Na+ is replaced by TEA+ and the internal K+ by Cs+, conditions
under which Na+- and K+-currents are blocked.
The BHK cells sealed in a similar manner to that observed before for many other celllines on the Patchliner© or Port-a-Patch©. Seals steadily increased during the sealing
procedure. However, these cells had fragile membranes and the whole cell configuration
was often established before a giga seal had been reached. Nevertheless, by the time
recordings were started, giga seals had formed (Na/K: 1.7 GΩ ± 0.7 GΩ, n = 4; TEA/Cs:3.1 GΩ ± 0.8 GΩ, n = 4). Current responses to the initial voltage ramp (-100 mV to+100 mV) of individual cells under the two different conditions (Na/K and TEA/Cs) areshown in Fig 1A. Initial currents at +95 mV are small (Na/K: 139 pA ± 23 pA, n = 4,Page 4 of 9

TEA/Cs: 325 pA ± 89 pA, n = 4). These small currents indicate that, if at all, only verysmall K+-currents are present.
The current response of the cell under conditions where Na+- and K+-currents can beobserved shows a characteristic Na+ current “hump” (4/4, Fig. 1A). This was notobserved in Na+-free (TEA/Cs) conditions (4/4) supporting the hypothesis that the humpis caused by Na+-flux. Running a Na+-current voltage protocol (voltage step from aholding potential of -80 mV to potentials between -60 mV and +60 mV for 20 ms inincrements of 10 mV, and then back to -80 mV) revealed typical Na+ current responses
Fig. 1B (n = 1).
To test what effect forskolin has on currents under the different conditions, voltage ramps
were applied every 5 s for a 5 min control period, once the whole cell configuration had
been established. Then 2 µM forskolin was added and currents were observed for anotherFigure 1: Untransfected BHK cells have Na+-currents but no forskolin regulatedcurrents. (A) Current responses (top) to a voltage ramp (bottom) of two individual
cells recorded in different conditions (TEA/Cs: Na+- and K+-free solutions, Na/K
standard solutions internal standard(F-), standard external). (B) Current responseof the cell shown in (A, Na/K) to a Na+-current pulse protocol. (C) Mean currents
at +95 mV for cells recorded under Na/K conditions (open bars, n = 4) and TEA/Cs
conditions (grey bars, n = 4).

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5 min before they were evaluated for the effect of forskolin. Currents under neither
condition changed significantly during the 5 min control period or within 5 min following
exposure to forskolin (Fig. 1C).
Sealing behavior and currents of BHK cells expressing wt CFTR
BHK cells expressing wt CFTR were handled according to the same protocols as theuntransfected BHK cells. As with the untransfected cells, membranes were fragile and thewhole cell configuration was established early in the sealing process, before giga seals
were formed. Seals increased steadily as long as the cells remained intact. However, once
the whole cell configuration was established, seals of the transfected cells appeared tosteadily decline.
Seal resistances, as determined from the current response to a 10mV voltage step from-80mV at the point in time when the first ramp protocol was executed, differedsignificantly between cells exposed to internal standard(F-) (150 MΩ ± 27 MΩ, n = 12)
and standard(Cl-) (650 MΩ ± 257 MΩ, n = 3). This difference in seal resistance is asurprising result since cells recorded with a F- containing internal solution usually reachat least the same seal resistance as cells recorded in internal solution without F-, as shownfor the wild-type BHK. For this reason, it is likely that the lower seal resistances are due
to open CFTR channels. CFTR channels are not voltage-activated. The calculation of theseal resistance would drop notedly by activation of CFTR channels in the whole cell
Figure 2: Initial current
responses are bigger forcells recorded in thepresence of internal Fthan
with
F--freesolutions. Shown are
current responses (top)to the initial voltage
ramp (bottom) for twocells, one recorded withinternalstandard(F-)
solution (F ) and one
withinternalstandard(Cl ) solution(Cl-).

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configuration. Fig. 2 shows whole cell currents of BHK cells expressing CFTR, one in
the presence of internal Cl- and one in the presence of internal F-. This difference incurrent amplitude can not be seen in wild-type BHK (Fig. 1). If this difference is due toactivation of CFTR by F- through the PKA pathway, this effect should not be further
enhanced by forskolin.
Fig. 3 shows currents from two individual cells recorded in the presence and absence ofinternal F-. Currents in the presence of internal F- increased with time (Fig. 3A & B)
whereas currents recorded in the absence of internal F- did not change significantly
within 5 minutes of recording (Fig. 2A & B). Exposure to 2 µM forskolin 5 min after
current recordings in the whole cell configuration had been started did not lead to asignificant change in the currents recorded with internal standard(F-) solution. Forskolin
activated currents recorded with internal standard(Cl-) (Fig. 3A & B). The reason that thedifference is not statistically significant is due to the low n number (n = 3), and thevariability in the responses. The concentration of 2µM forskolin is not fully activating(Seamon, Padgett, and Daly, 1981). Interestingly, currents recorded in the presence andFigure 3: Forskolin does notactivate currents further whenchannels are already activated byF-. (A) shows the current
responses at +95 mV for two
individual cells, one recorded
withinternalstandard(F-)
solution (F ) and one withstandard(Cl-) solution (Cl-). (B)Mean currens at +95 mV for cells
recorded in the presence ofinternal F- (open bars, n = 12) andin the absence of internal F- (greybars, n = 3). * denotes significant
difference to data obtained under
the same conditions at control. #denotes significant difference todata obtained under different
conditions (internal F- vs. Cl-).

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absence of internal F- did not differ significantly from each other after 5 min exposure toforskolin (Fig. 3B). However, due to the different internal Cl- concentrations it is not agiven that channel activity is the same under both conditions.

Discussion
In this report we summarize some preliminary experiments investigating the effect of
intracellular F- on CFTR currents measured in the whole cell configuration on thePatchliner©. This is by no means meant to be a complete study.
In summary, our measurements show that there is an activation of CFTR channels by Fwhich cannot be further enhanced by forskolin. Looking at the literature, one explanation
for this finding seems very plausible: F- interferes in the same step as forskolin within thesignaling pathway (Fig. 4), although via a different mechanism, both F- and forskolin
activate adenylate cyclase (Eckstein et al., 1979, Insel and Ostrom, 2003). Once
activated, the cyclase converts ATP into cAMP. This leads to phosphorylation of protein
kinase A (PKA) and the kinase becomes active. The active kinase can then phosphorylate
the CFTR, openings of which are then controlled by ATP binding to the cytoplasmic
nucleotide binding site.

Figure 4: Scheme for the signalling pathway of phosphorylation of CFTR byforskolin and fluoride.

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In conclusion, the findings reported in this study suggest that internal F- activates CFTR
by upregulation of adenylate cyclase. This raises the possibility of replacing expensive
activator forskolin with internal F- for CFTR studies. To confirm this will require furtherinvestigation.

Literature
Zhou, Z., Wang, X., Liu, H., Zou, X., Li, M. Hwang, T. (2006) The two ATP bindingsites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct rolesin the gating kinetics and energetics. J Gen Physiol 128(4): 413-422
Ecksteine, F., Cassel, D., Levkovitz, H., Lowe, M., Selinger, Z. (1979) Guanosine 5’-O(2-Thiodiphosphate) An inhibitor of adenylated cyclase stimulation by guanine
nucleotides and fluoride ions. J Biol Chem 254(10): 9829-1834Insel, P.A. and Ostrom, R.S. (2003) Forskolin as a Tool for Examining Adenylyl CyclaseExpression, Regulation, and G Protein Signaling Cellular and Molecular Neurobiology
23(3): 305-314
Seamon, K.B., Padgett,W., Daly, J.W. (1981) Forskolin: Unique diterpene activator ofadenylate cyclase in membranes and in intact cells, Proc. NatL Acad. Sci. USA
78(6):3363-3367

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