EC18 as a Tool to Understand the Role of HCN4 Channels in Mediating Hyperpolarization-Activated Current in Tissues

Article abstract of DOI:10.1021/acsmedchemlett.8b00587

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are membrane proteins encoded by four genes (HCN1-4) and widely distributed in the central and peripheral nervous system and in the heart. HCN channels are involved in several physiological functions, including the generation of rhythmic activity, and are considered important drug targets if compounds with isoform selectivity are developed. At present, however, few compounds are known, which are able to discriminate among HCN channel isoforms. The inclusion of the three-methylene chain of zatebradine into a cyclohexane ring gave a compound (3a) showing a 5-fold preference for HCN4 channels, and ability to selectively modulate I_h in different tissues. Compound 3a has been tested for its ability to reduce I_h and to interact with other ion channels in the heart and the central nervous system. Its preference for HCN4 channels makes this compound useful to elucidate the contribution of this isoform in the physiological and pathological processes involving hyperpolarization-activated current.

Full text of DOI:10.1021/acsmedchemlett.8b00587

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
EC18 as a Tool To Understand the Role of HCN4 Channels in
Mediating Hyperpolarization-Activated Current in Tissues
,
†
†
†,∥
‡
Maria Novella Romanelli,* Martina Del Lungo, Luca Guandalini, Mehrnoush Zobeiri,
§
§
§
†
†
́
Gyokeres, Tamas Arpadffy-Lovas, Istvan Koncz, Laura Sartiani, Gianluca Bartolucci,
̈
́ ́ ́
Andras
Silvia Dei, Dina Manetti, Elisabetta Teodori, Thomas Budde, and Elisabetta Cerbai
†
†
†
‡
†
†Department of Neurosciences, Psychology, Drug Research and Child Health (NeuroFarBa), University of Florence, Florence
5
0139, Italy
‡
Institute of Physiology I, Westfal
§
̈
ische Wilhelms-University Mu
Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged H-6720, Hungary
S Supporting Information
nster, Munster 48149, Germany
̈ ̈
*
ABSTRACT: Hyperpolarization-activated cyclic nucleotide-
gated (HCN) channels are membrane proteins encoded by
four genes (HCN1−4) and widely distributed in the central
and peripheral nervous system and in the heart. HCN
channels are involved in several physiological functions,
including the generation of rhythmic activity, and are
considered important drug targets if compounds with isoform
selectivity are developed. At present, however, few compounds
are known, which are able to discriminate among HCN channel isoforms. The inclusion of the three-methylene chain of
zatebradine into a cyclohexane ring gave a compound (3a) showing a 5-fold preference for HCN4 channels, and ability to
selectively modulate I in different tissues. Compound 3a has been tested for its ability to reduce I and to interact with other
h
h
ion channels in the heart and the central nervous system. Its preference for HCN4 channels makes this compound useful to
elucidate the contribution of this isoform in the physiological and pathological processes involving hyperpolarization-activated
current.
KEYWORDS: Hyperpolarization-activated current, HCN channels, isoform selectivity, phenylalkylamines, zatebradine analogues,
rigid analogues
yperpolarization-activated cyclic nucleotide-gated
HCN1 and HCN2 are more widely distributed than HCN4,
which is mainly located in the thalamus. In the retina, where
5
H
(HCN) channels are members of the superfamilies of
+
voltage-dependent K (K ) and cyclic nucleotide-gated (CNG)
HCN channels shape the light response, all four isoform are
v
6
channels. The architecture of these proteins resembles that of
voltage-gated potassium channels, showing a tetrameric
arrangement, each subunit being composed of six trans-
membrane domains including the voltage sensor S4, and a pore
expressed. In DRG, HCN1 and HCN2 are the most abundant
7
isoforms.
HCN channels can be considered important drug targets for
cardiac pathologies and neurological diseases such as pain and
8
,9
loop between S5 and S6. At variance with K channels, the C-
epilepsy, but the modulation of these channels has been
exploited so far only at the cardiac level. Ivabradine (Figure 1),
the only drug developed as a specific HCN channel blocker,
has been approved as a bradycardic agent in chronic stable
angina pectoris and to reduce hospitalization from worsening
v
terminal intracellular portion harbors a Cyclic Nucleotide
+
Binding Domain (CNBD). HCN channels carry a mixed Na /
+
K current known as hyperpolarization-activated current (I ),
h
are activated at hyperpolarizing voltages, and are modulated by
cyclic nucleotides (cAMP, cGMP, and others). Four isoforms
have been cloned (HCN1−4), differing in localization and
1
0,11
heart failure.
suggested a role of HCN channels in pain perception and in
In addition, several lines of evidence have
1
−3
9
,12−14
biophysical properties.
epilepsy.
Indeed, ivabradine is able to attenuate
HCN channels are involved in various physiological
15,16
neuropathic and inflammatory pain
threshold for maximal electroshock-induced seizures. How-
and to increase the
processes in the central and peripheral nervous systems and
17
1
in non-neuronal districts. For instance, HCN channels control
heart pace in the sino-atrial node (SAN), where HCN4 is the
most abundant isoform. In the central nervous system (CNS),
4
Special Issue: Highlighting Medicinal Chemistry in Italy
HCN channels regulate several functions, among which are
intrinsic excitability and rhythmogenesis. All four isoforms are
expressed, to different extent and with different distribution:
Received: November 29, 2018
Accepted: February 6, 2019
Published: February 6, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00587
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Compounds 3a−g
Figure 1. Structure of ivabradine, zatebradine, and analogues 1, 2, and
3a endowed with isoform preference or selectivity.
a
Reagents and conditions: (a) (1) NBS, AIBN, CCl , heating; (2) t-
4
BuOK, 7,8-dimethoxy-2,3-dihydro-1H-3-benzazepin-2-one; (b) H2/
Pd/C; (c) chromatographic separation; (d) NaOH; (e) DPPA,
ever, although ivabradine is well tolerated, its use for new
therapeutic applications may be restricted due to its lack of
selectivity since it blocks the four human HCN isoforms with
toluene, then HCl; (f) (1) SOCl , (2) NaN , acetone; (3) Δ, toluene;
2
3
1
8
(4) HCl; (g) arylalkyl halides 8a−e, Et
3
N; (h) CH O, HCOOH. Ar =
,4-dimethoxyphenyl. X = (CH ) (a); CH2 (b); (CH2) (c);
2
similar potency. Several clinically used drugs have been
3
19
2
2
3
reported to modulate Ih, with few of them showing some
(
CH ) O (d); CH CHCH (e); 3f, R = 3,4-dimethoxybenzyl, X =
2
0
2
2
2
isoform selectivity, such as the anesthetic propofol for HCN1
CH .
2
21
or the anticonvulsant gabapentin for HCN4. However, these
molecules are not specific for HCN channels and interact with
other targets, while isoform-selective inhibitors could have a
safer pharmacological profile due to the reduction of side
effects.
Table 1. Percentage of Reduction of I at −80 mV after
h
Application of Drugs at the Concentration of 5 μM on
a
Human HCN4 Channel Expressed in HEK 293 Cells
Recently some of us have described the design, synthesis,
and characterization of phenylalkylamines endowed with
isoform preference or selectivity (Figure 1). In HEK 293
cells stably expressing homomeric HCN1, HCN2, and HCN4
channels, MEL57A (1) selectively inhibited the HCN1
N
R1
Me
R2
Ih reduction (%)
2
2,23
isoform.
in native tissues, where the channels may exist in the
heteromeric form. Compound 1 was able to reduce I in
Importantly, this selectivity was also maintained
b
3
(
a (EC18)
+)-3a
(−)-3a
−CH CH Ar
59 ± 14 (n = 5)
46 ± 7.7 (n = 3)
45 ± 20.1 (n = 4)
21 ± 12 (n = 6)
47 ± 16.1 (n = 4)
48 ± 6.9 (n = 7)
64 ± 8.0 (n = 4)
∼4 (n = 4)
2
2
Me
Me
Me
Me
Me
Me
−CH CH Ar
2 2
h
−CH CH Ar
2
2
23
DRG neurons at doses that did not influence heart rate;
accordingly, 1 was effective in reducing hyperalgesia and
b
3
3
3
3
3
3
b
c
d
e
f
g
−CH Ar
2
b
−CH CH CH Ar
2 2 2
24
b
allodynia in oxaliplatin-treated rats without cardiac effects.
−CH CH OAr
2 2
b
Compound 2 (MEL55A) is an example of isoform-preferring
−CH CHCHAr
2
b
compound since it was shown to preferentially block HCN1
CH Ar
−CH Ar
2
2
22
c
and HCN2 over HCN4; as its dimeric analogue 1, it was able
Me
−CH CH Ar
30 ± 11.6 (n = 4)
2
2
25
d
to relieve chemotherapy-induced neuropathic pain. In this
Letter we describe the design, synthesis, and characterization
zatebradine
59 ± 19 (n = 5)
a
Values are normalized to control and represent the mean ± SD of
2
3
of 3a (EC18), a phenylalkylamine showing preference for
HCN4 channels, and its usefulness to evaluate the physio-
logical role of this isoform.
b
c
3
−7 experiments. Single bond. Double bond on both rings.
d
Obtained with a 10 μM concentration. Ar = 3,4-dimethoxyphenyl.
Compound 3a (EC18) represents a rigid analogue of
zatebradine (Figure 1), the first “specific bradycardic agent”
we thought it would be important to also synthesize 3g, the
unsaturated analogue of 3a, since the presence of double bonds
gave interesting properties to 1 and 2.
19
that entered clinical trials. The inclusion of the three-
methylene chain into a cyclohexane ring sharply reduced
zatebradine’s flexibility, as 3a is able to adopt different
conformations only in the phenethyl moiety. By this way it
was envisaged that some selectivity could be obtained; a similar
modification made on verapamil, a structural analogue of
zatebradine, caused a separation of vasodilator and cardiac
The synthesis of compounds 3a−g was performed as shown
27
in Scheme 1. Following the procedure reported by Allan,
methyl cyclohex-3-ene-1-carboxylate was reacted first with N-
bromosuccinimide and then with the potassium salt of 7,8-
2
8
dimethoxy-2,3-dihydro-1H-3-benzazepin-2-one, giving 4,
which was hydrogenated on Pd/C yielding ester 5. This
preparation was performed several times, obtaining 5 with
similar yields (20−25%) and as mixtures of isomers, the most
abundant one being the cis ester (85−95%), which was
26
depressant activities. A small set of cyclohexane derivatives
(
3a−f, see structure in Scheme 1 and Table 1), characterized
by different spacers between the basic nitrogen and the
dimethoxyphenyl ring, was designed and prepared. In addition,
B
DOI: 10.1021/acsmedchemlett.8b00587
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ACS Medicinal Chemistry Letters
Letter
separated from the trans one by means of chromatography.
The cis−trans configuration was assessed by means of NMR, as
finding in line with the low expression of this isoform in DRG
neurons.
25
26
previously done for other cyclohexane derivatives: the width
of the peak related to the cyclohexyl proton in position 1 was
wider for the cis isomer (H axial) with respect to the trans one
EC18 (3a) represents the first HCN4-preferring compound,
discovered in a project aimed to find zatebradine analogues
22,23
endowed with isoform selectivity.
In fact, when tested on a
(
H equatorial). The cis configuration was then confirmed by a
recombinant HCN4 channel, EC18 showed a potency in the
same range as ivabradine, but a 5-fold preference for this
isoform with respect to HCN1 and HCN2, while ivabradine
reduced h-current with the same potency on the three
NOESY experiment. Alkaline hydrolysis of cis-5 gave the
corresponding acid 6, which was transformed into amine 7
through Curtius rearrangement of the acyl azide. This step was
performed in two different ways. Treatment of the acid with
DPPA (diphenyl phosphoryl azide) in toluene (method A),
followed by acid hydrolysis of the intermediate isocyanate,
gave 7 in about 50% yield. Alternatively, the acyl azide was
prepared by reaction of the acyl chloride with sodium azide;
heating in toluene and subsequent acid hydrolysis gave the
amine 7 in slightly higher yields (method B, 60% yield).
Reaction of 7 with the suitable arylalkyl halide (8a−e) gave
secondary amines 9a−e, which were transformed into N-
methyl-derivatives 3a−e by reaction with formaldehyde and
formic acid; when using 3,4-dimethoxybenzyl bromide, some
amount (4%) of double addition product 3f was obtained.
Compound 4 was transformed into 3g using the same reaction
sequence but avoiding catalytic hydrogenation.
18
isoforms. Notably, the preference obtained at recombinant
channels is maintained in native tissues, thus making 3a a tool
to study the individual function of HCN4 channels in cardiac
cells and neurons. Other HCN4-selective/preferring com-
pounds have been described, such as the already mentioned
21
30
gabapentin and amiodarone. However, both compounds
have been developed for interaction with other targets, and
their multi-ion channels blocking properties can obscure
selectivity for HCN4 channels, even if the activity on HCN4
channel may contribute to their pharmacological effect.
Although a complete characterization of 3a on other
channels or receptors has not been done yet, some experiments
on cardiac tissue suggest that 3a also interacts with other ion
channels. In fact, in dog ventricular papillary muscle at a
stimulation cycle length of 1000 ms, 3a (10 μM) significantly
and strongly inhibited the Vmax and increased conduction time
(Figure 2, Table S1). Compound 3a moderately shortened the
action potential (AP) duration at 1 μM, then prolonged the
action potential duration at 10 μM.
As a preliminary screening, the compounds were tested at a
μM concentration to check their ability to block h-current on
5
human HCN4 stably expressed in HEK 293 cells, at voltages
(
(
−80 mV) mimicking physiological membrane potential
Table 1). Under these conditions all the compounds were
able to inhibit h-current, the majority of the compounds
producing about 50% reduction. Representative traces are
reported in Figure S1. The benzyl analogue 3b and the
unsaturated derivative 3g were much less active, the extent of
reduction being smaller (21% and 30%, respectively); the
dibenzyl compound 3f was almost inactive. To understand the
importance of chirality, the enantiomers of 3a were separated
by means of HPLC (Figures S2 and S3), but no
enantioselectivity was observed (Table 1). Although small,
this set of compounds can suggest some structure−activity
relationships: the insertion of two double bonds in the
azepinone and cyclohexane rings is not productive, as well as
the increase in size of both R and R substituents. On the
1
2
contrary, the length and type of the spacer between the basic N
atom and the dimethoxyphenyl moiety can be varied, still
maintaining good potency.
Owing to its similarity with zatebradine, compound 3a
(
EC18) was selected for further characterization, which has
23
been partially disclosed previously. This compound was
tested on the homomeric HCN1, HCN2, and HCN4 channels
expressed in HEK293 cells: it showed a 5-fold preference for
HCN4 (EC50 3.98 ± 1.16 μM) over HCN1 (EC50 21 ± 3.98
μM) and HCN2 (EC50 19.35 ± 4.48 μM). Consistently with
its good activity on HCN4, in guinea pig SAN 10 μM 3a
Figure 2. Effect of 3a (EC18) on the action potential repolarization in
dog papillary muscle (n = 5) at stimulation cycle length of 1000 ms
(representative action potential waveform recordings originate from
the same preparation).
reduced I by ∼67% at −120 mV, and on dog cardiac Purkinje
EC18 was found to have a cardiac electrophysiological
profile similar to that of ivabradine in view of the ability to
inhibit Vmax 3and to prolong repolarization at 10 μM
h
fibers it reduced the amplitude and slowed the slope of
23
diastolic depolarization phase. Moreover, on guinea-pig
spontaneously beating isolated atria, it produced a negative
chronotropic effect (IC50 9.9 ± 5.5 μM), with potency
1
concentration. The AP duration shortening exerted by 1
+
μM EC18 is probably attributable to Na channel blockade.
2
9
+
comparable to zatebradine (IC50 13.7 ± 8.7 μM).
The inhibitory effect of EC18 on Na channels might still exist
In line with its lower activity on HCN1 and HCN2 channels,
at 10 μM, but regarding the effect of EC18 on the
+
on mouse DRG neurons 3a did not affect I conductance, at a
repolarization, the simultaneous K channel blockade could
h
physiologically relevant voltage, up to a concentration of 100
μM. Indeed, 3a was unable to reduce hypersensitivity when
outweigh this AP shortening effect. Further studies are
required for establishing the degree of the K current inhibition
2
3
+
tested in a mouse model of oxaliplatin-induced neuropathy, a
by EC18 in cardiomyocytes. Ivabradine, in addition to blocking
C
DOI: 10.1021/acsmedchemlett.8b00587
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
the pacemaker current, was found able to inhibit hERG
channels with a potency that is similar to that reported for
HCN4 (see ref 1 and references therein). EC18 might have a
similar action.
EC18 can be useful to study HCN channels not only in
heart but also in the CNS. In the thalamus, a brain region
33,34
mainly expressing HCN4 and HCN2 channels,
compound
3
a has been used to understand the contribution of the HCN4
isoform to I in small and large interneurons of the dorsal part
h
of the lateral geniculate nucleus, which are classified based on
differences in I current density. In these GABAergic neurons
h
35
3
0 μM 3a reduced I by about 31.4 ± 5.2%. As a further
h
characterization, in this work we report the effect of 3a in
thalamocortical relay (TC) neurons of the ventrobasal
thalamic complex (VB), which belong to the principal thalamic
neurons. Here, compound 3a revealed stronger effects with 30
μM blocking 91.3 ± 0.7% (n = 2) of I in these cells (Figure
h
3
A). The construction of a dose−response relationship
+
Figure 4. Effect of compound 3a on K outward currents in thalamic
neurons. (A) Families of outward currents evoked by standard voltage
protocols (see lower inset and Supporting Information 1) under
control conditions (black traces) and in the presence of 10 μM 3a
33
(
(
red traces) following acute isolation. Late current components
arrowhead) but not the early transient currents (arrow) were
affected. The 3a-sensitive component is shown in the upper inset
(
scale bars represent 200 ms and 500 pA). (B) Current−voltage
relationship of the late current component (see arrowhead in A).
The data reported in this Letter suggest that compound 3a is
a valuable tool to study HCN channel isoforms in the CNS
and in heart, useful to clarify the contribution of different
HCN isoforms in mediating I . As well as for compounds 1
h
and 2, the ability of 3a to discriminate among HCN channel
isoforms is an advantage with respect to compounds such as
ZD7288 or ivabradine, which are commonly used to block Ih
but are not isoform-selective and, as well as 3a, nor absolutely
Figure 3. Effect of compound 3a on Ih in thalamic neurons. (A)
Sample whole-cell patch clamp recordings showing the current
32
reduction in VB TC neurons in brain slices by different 3a
concentrations (1, 10, and 30 μM as indicated). I in the presence
h
37,38
specific for HCN channels.
(
red line) and absence (black lines) of 3a was elicited by standard
In conclusion, a compound has been discovered, 3a (EC18),
showing some selectivity for HCN4 channels, and able to
discriminate among HCN channel isoform in recombinant
systems and in native tissues. Even if 3a shows some activity on
Na and K currents, its HCN4 blocking properties make it a
valuable tool to study HCN4 channels in a physiological
environment or in pathologies, such as those of the CNS. In
particular, the HCN4-blocking activity in the thalamus opens
the way to test this compound in animal models of
neurological disorders; the results of these tests will be
presented in due time. Work is underway to improve potency
and selectivity of the lead compound.
voltage protocols (see Supporting Information 1). Scale bars
represent 1 nA and 2 s. (B) Dose−response relationship of I
h
amplitude reduction. The EC value was estimated by fitting a
5
0
sigmoidal function (red line) to the data points (see Supporting
Information 1).
+
+
pointed to an EC50 value of around 11 μM (Figure 3B).
These data suggest that in TC neurons I is mainly mediated
h
by HCN4 or HCN2/4 heteromers and indicate that
compound 3a is a valuable tool for analyzing the cell type-
specific properties and function of HCN channels in native
neurons.
+
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00587.
An effect on K current was found also in VB TC neurons
Figure 4). When K outward currents were induced by step
■
+
*
S
(
depolarizations (Figure 4A, lower inset) 10 μM 3a blocked
4.5 ± 2.2% (n = 8; Figure 4B) of the late component while
1
leaving the early transient current unaffected. These findings
indicate that compound 3a has selective effects on different
Synthesis and characterization of compounds, chiral
separation, pharmacological methods on HEK-293 cells,
+
types of voltage-dependent K channels, blocking slow delayed
+
1
13
rectifier channels (Figure 4A upper inset) but not affecting K
channels carrying A-type current in TC neurons.
heart, and CNS; H and C NMR spectra, and LC-
DAD analysis (PDF)
32,36
D
DOI: 10.1021/acsmedchemlett.8b00587
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
H and 13C NMR spectra, and LC- DAD analysis
Letter
1
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(9) DiFrancesco, J. C.; DiFrancesco, D. Dysfunctional HCN ion
channels in neurological diseases. Front. Cell. Neurosci. 2015, 9, 71.
(PDF)
AUTHOR INFORMATION
Corresponding Author
Phone 0039 055 4573691. E-mail novella.romanelli@unifi.it.
■
*
ORCID
(
10) Sulfi, S.; Timmis, A. D. Ivabradine − the first selective sinus
Maria Novella Romanelli: 0000-0002-5685-3403
Gianluca Bartolucci: 0000-0002-5631-8769
Dina Manetti: 0000-0002-5881-6550
Present Address
node If channel inhibitor in the treatment of stable angina. Int. J.
Clin.Pract. 2006, 60, 222−228.
(11) Bielecka-Dabrowa, A.; von Haehling, S.; Rysz, J.; Banach, M.
Novel drugs for heart rate control in heart failure. Heart Failure Rev.
2018, 23 (4), 517−525.
∥
Stabilimento Chimico Farmaceutico Militare, Ministero della
(
12) Emery, E. C.; Young, G. T.; McNaughton, P. A. HCN2 ion
Difesa, Via Reginaldo Giuliani 201, I-50141 Firenze, Italy.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
channels: an emerging role as the pacemakers of pain. Trends
Pharmacol. Sci. 2012, 33 (8), 456−463.
(13) Zhang, H.; Dougherty, P. M. Enhanced Excitability of Primary
Sensory Neurons and Altered Gene Expression of Neuronal Ion
Channels in Dorsal Root Ganglion in Paclitaxel-induced Peripheral
Neuropathy. Anesthesiology 2014, 120 (6), 1463−1475.
Funding
(
14) Campostrini, G.; DiFrancesco, J. C.; Castellotti, B.; Milanesi,
M.N.R., M.D.L., L.G., L.S., and E.C. have been supported by
Normacor (Contract LSH M/CT/2006/018676). T.B. was
supported by DFG (BU 1019/15-1/16-1). A.G., T.A.L., and
I.K. were supported by the Hungarian Government-Ministry of
Human Resources (grant EFOP-3.6.2-16-2017-00006, LIVE
LONGER).
R.; Gnecchi-Ruscone, T.; Bonzanni, M.; Bucchi, A.; Baruscotti, M.;
Ferrarese, C.; Franceschetti, S.; Canafoglia, L.; Ragona, F.; Freri, E.;
Labate, A.; Gambardella, A.; Costa, C.; Gellera, C.; Granata, T.;
Barbuti, A.; DiFrancesco, D. A Loss-of-Function HCN4 Mutation
Associated With Familial Benign Myoclonic Epilepsy in Infancy
Causes Increased Neuronal Excitability. Front. Mol. Neurosci. 2018,
1
(
1, 269.
Notes
15) Descoeur, J.; Pereira, V.; Pizzoccaro, A.; Francois, A.; Ling, B.;
The authors declare no competing financial interest.
Maffre, V.; Couette, B.; Busserolles, J.; Courteix, C.; Noel, J.;
Lazdunski, M.; Eschalier, A.; Authier, N.; Bourinet, E. Oxaliplatin-
induced cold hypersensitivity is due to remodelling of ion channel
expression in nociceptors. EMBO Mol. Med. 2011, 3 (5), 266−278.
ACKNOWLEDGMENTS
■
We want to thank Prof. Martin Biel (University of Munich,
Germany) for providing stably transfected HCN1, HCN2, and
HCN4 HEK293 cells. We wish to thank Prof. Andras Varro
(
16) Young, G. T.; Emery, E. C.; Mooney, E. R.; Tsantoulas, C.;
̀
McNaughton, P. A. Inflammatory and neuropathic pain are rapidly
suppressed by peripheral block of hyperpolarisation-activated cyclic
nucleotide-gated ion channels. Pain 2014, 155, 1708−1719.
(
(
University of Szeged) and Prof. Alessandro Mugelli
University of Florence) for scientific support.
(
17) Luszczki, J. J.; Prystupa, A.; Andres-Mach, M.; Marzeda, E.;
̂
Florek-Luszczki, M. Ivabradine (a hyperpolarization activated cyclic
nucleotide-gated channel blocker) elevates the threshold for maximal
electroshock-induced tonic seizures in mice. Pharmacol. Rep. 2013, 65,
ABBREVIATIONS
■
AP, action potential; HCN, hyperpolarization-activated cyclic
nucleotide-gated; DPPA, diphenyl phosphoryl azide; DRG,
dorsal root ganglion; SAN, sino-atrial node; TC, thalamocort-
ical relay neurons; VB, ventrobasal thalamic complex
1
(
407−1414.
18) Stieber, J.; Wieland, K.; Stockl, G.; Ludwig, A.; Hofmann, F.
Bradycardic and Proarrhythmic Properties of Sinus Node Inhibitors.
Mol. Pharmacol. 2006, 69, 1328−1337.
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