Α-Dendrotoxin Inhibits the ASIC Current in Dorsal Root Ganglion

Home , Dendrotoxin

Neuroscience Letters 606 (2015) 42–47

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Neuroscience Letters

journal homepage: www.elsevier.com/locate/neulet

Research paper

␣-Dendrotoxin inhibits the ASIC current in dorsal root ganglion neurons from rat

a a b c b

Adriana Báez , Emilio Salceda , Martín Fló , Martín Grana˜ , Cecilia Fernández ,

a a,∗

Rosario Vega , Enrique Soto

Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla (BUAP), 14 sur 6301, CU, San Manuel, Puebla, Pue., CP 72570, Mexico

Faculty of Chemistry, UDELAR, Av. Gral., Flores, Montevideo 2124, Uruguay

Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay

h i g h l i g h t s g r a p h i c a l a b s t r a c t

•

␣-DTx inhibits the peak amplitude of

proton gated current (ASIC) in DRG neurons.

•

␣-DTx action on ASICs is reversible

and dose dependent.

•

␣-DTx inhibits ASIC currents with

IC50 in the nM range.

•

␣-DTx positive charge at neutral pH is

similar to other components inhibit-

ing ASICs.

•

Other toxins considered to be highly

selective may have dual action such

as ␣-DTx.

a r t i c l e i n f o a b s t r a c t

Article history: Dendrotoxins are a group of peptide toxins puriﬁed from the venom of several mamba snakes. ␣-

Received 27 April 2015

Dendrotoxin (␣-DTx, from the Eastern green mamba Dendroaspis angusticeps) is a well-known blocker

Received in revised form 11 August 2015 +

of voltage-gated K channels and speciﬁcally of Kv1.1, Kv1.2 and Kv1.6. In this work we show that ␣-

Accepted 19 August 2015

DTx inhibited the ASIC currents in DRG neurons (IC50 = 0.8 ␮M) when continuously perfused during 25 s

Available online 24 August 2015

(including a 5 s pulse to pH 6.1), but not when co-applied with the pH drop. Additionally, we show that

␣

-DTx abolished a transient component of the outward current that, in some experiments, appeared

Keywords:

immediately after the end of the acid pulse. Our data indicate that ␣-DTx inhibits ASICs in the high nM

Kunitz domain

␣

ASIC range while some Kv are inhibited in the low nM range. The -DTx selectivity and its potential interaction

APETx2 with ASICs should be taken in consideration when DTx is used in the high nM range.

KNa

Proton gated

http://dx.doi.org/10.1016/j.neulet.2015.08.034

A. Báez et al. / Neuroscience Letters 606 (2015) 42–47 43

1. Introduction plated on 12 mm × 10 mm glass coverslips (Corning, Corning, NY)

pretreated with poly-d-lysine (Sigma–Aldrich) and placed onto

Acid-sensing ion channels (ASICs) are membrane proteins 35 mm culture dishes (Corning). Neurons were incubated 2–8 h in

◦

widely expressed in neurons of central (CNS) and peripheral ner- a humidiﬁed atmosphere (95% air, 5% CO2, at 37 C) using a CO2

vous system and also in non-neuronal tissues. ASICs constitute a water-jacketed incubator (Nuaire, Plymouth, MN) to allow them

group of sodium-selective channels that are activated by extracel- to settle and adhere to the coverslips. The incubation medium (pH

lular acidosis. They belong to the ENaC/Degenerin family and share 7.4) contained L15, 15.7 mM NaHCO3 (Merck, Naucalpan, Mexico),

three main features with other members of the family: sodium per- 10% fetal bovine serum, 2.5 ␮g/mL fungizone (both from Invitro-

meability, amiloride sensitivity and insensitivity to voltage changes gen), 100 U/mL penicillin (Lakeside, Toluca, Mexico), and 15.8 mM

[1–4]. HEPES (Sigma–Aldrich).

ASICs are involved in pathological and physiological processes.

In the periphery, they participate in various sensory modalities,

mechanoreception and nociception, touch and taste perception, 2.2. Electrophysiological recording

in the auditory and vestibular systems [5,6], as well as in cardiac

ischemic pain [7]. In the CNS, ASICs participate in synaptic plas- A coverslip with attached cells was transferred to a 500 ␮L per-

ticity, learning and memory, and in the physiopathology of brain fusion chamber mounted on the stage of an inverted phase-contrast

ischemia among other processes [8]. ASIC channels are homo or microscope (TMS, Nikon Co., Tokyo, Japan). Cells were bathed with

◦

heterotrimers of six subunits encoded by four genes, ACCN1-4; the external solution (24 C) of the following composition (in mM):

these give rise to ASIC1a, 1b, 2a, 2b, 3 and 4, where a and b are alter- NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.2, HEPES 10 (Extracellular

native splice variants that yield proteins with distinct N-termini [3]. pH 7.4 or 6.1 was adjusted with NaOH; MES instead of HEPES

Except for ASIC4, the remaining subunits are expressed in primary was used for solution with pH 6.1). Current recording was per-

sensory neurons of the trigeminal, vagal, and dorsal root ganglia formed using an Axopatch 1D ampliﬁer (Molecular Devices, Union

(DRG) [9]. City, CA). Command-pulse generation and data sampling were con-

␣ ␣

-Dendrotoxin ( -DTx) is a well-known blocker of voltage- trolled by pClamp 9.2 software (Molecular Devices) using a 16-bit

gated K channels, speciﬁcally of Kv1.1, Kv1.2 and Kv1.6 [10–12]. data-acquisition system (Digidata 1320, Molecular Devices). Sig-

It is a very basic polypeptide puriﬁed from the venom of the East- nals were low-pass ﬁltered at 5 kHz and digitized at 5 kHz. Patch

␣

ern green mamba (Dendroaspis angusticeps) [13]. -DTx contains 59 pipettes were pulled from borosilicate glass capillaries (TW120-3;

amino acids folded into a single Kunitz domain, i.e., a compact ˛ + ˇ WPI, Sarasota, FL) using a Flaming-Brown electrode puller (80-PC;

structure cross-linked by three disulﬁde bonds [11]. ASICs have Sutter Instruments Company, San Rafael, CA). They typically had a

recently received quite a lot of attention due to their involvement in resistance of 1.5–2.5 M when ﬁlled with the intracellular solution

various physiological and pathological processes [4]. In the midst (in mM) NaCl 10, KCl 125, CaCl2 0.13, EGTA 10, HEPES 5, NaGTP 1,

of a research project on the functional characterization of Kunitz MgATP 2 (Intracellular pH 7.2 adjusted with KOH). The series resis-

inhibitors, we made the observation that, apart from its effect on K tance was electronically compensated (≈80%). In the time course of

channels, ␣-DTx produced a clear and consistent reduction of ASIC an experiment, seal and series resistance were continuously mon-

currents from DRG neurons; thence we decided to systematically itored to guarantee stable recording conditions. Recordings were

study the action of ␣-DTx on these currents. not included in the analysis if the access resistance changed >10%

during the experiment.

2. Materials and methods

2.3. Experimental protocols

To study the effect of ␣-DTx on the ASIC current, the whole

cell patch-clamp technique was used. For this purpose, the DRG

Cells were bath-perfused with extracellular solution at pH 7.4

neurons from Long Evans CII/ZV rats (P7–P10) of either sex were

employing a peristaltic pump (Masterﬂex, L/S Easy-Load II, Cole

isolated and cultured. Animal care and procedures were in accor-

Parmer, Vernon Hills, IL). ASIC currents were generated by a fast pH

dance with the National Institutes of Health Guide for the Care

change from 7.4 to 6.1 for 5 s, by shifting one of the three outlets

and Use of Laboratory Animals. All efforts were done to minimize

of a fast change perfusion system (SF-77B, Warner Inst., Hamden,

animal suffering and to reduce the number of animals used. Exper-

CT) while keeping the cell at a holding potential (V ) of −60 mV.

imental procedures were similar to those previously described by h

Time between individual sweeps was 1 min to guarantee complete

our research group [14].

recovery of ASIC from desensitization. Capsazepine (10 ␮M) was

added to the extracellular solution (pH 6.1) to avoid the activation

2.1. Cell culture

of the TRPV1 receptor present in DRG neurons. A pH of 6.1 was used

to activate ASIC currents because this is the pH at which half of the

Brieﬂy, the rats were anesthetized and killed with an over-

maximum current was attained (pH50 = 6 ± 0.1). The pKa value of

dose of sevoﬂuorane and subsequently decapitated. DRGs were

◦ the buffer employed in the acidic solution (MES) is 6.15. For each

isolated and incubated (30 min at 37 C) in Leibovitz L15 medium

experiment, at least two control responses were recorded before

(L15) (Invitrogen, Carlsbad, CA) containing 1.25 mg/mL trypsin and

any experimental manipulation. The current parameters measured

1.25 mg/mL collagenase (both from Sigma–Aldrich, St. Louis, MO,

to characterize the ASIC currents were: (a) the maximum peak

USA). After the enzyme treatment, the ganglia were washed three

amplitude (Ipeak), (b) the current desensitization time-constant

times with sterile L15 and they were mechanically dissociated

(des) obtained by ﬁtting a single exponential function to the desen-

using a Pasteur pipette. The cells obtained in this way were then

sitization phase of the current with, (c) the current amplitude at

the steady state (Iend) measured at the last 100 ms of the acid pH

pulse. ␣-DTx was applied 20 s before the acid pulse and during the ∗

Corresponding author.

whole 5 s acid pulse (sustained application); in some experiments,

E-mail addresses: [email protected] (A. Báez), [email protected]

the toxin was only applied during the 5 s acid pulse (co-application)

(E. Salceda), martinﬂ[email protected] (M. Fló), [email protected]

[14,15]. ␣-DTx was kindly donated by Dr. Carlos Cervenansky˜ from

(M. Grana),˜ [email protected] (C. Fernández), axolotl [email protected] (R. Vega),

[email protected] (E. Soto). the Analytical Biochemistry and Proteomics Unit, Institut Pasteur

44 A. Báez et al. / Neuroscience Letters 606 (2015) 42–47

de Montevideo/Instituto de Investigaciones Biológicas Clemente

Estable (Uruguay).

2.4. Data analysis

Data were analyzed off-line using Clampﬁt 10.2 (Molecular

Devices) and OriginPro 8 (OriginLab, Northampton, MA) software.

The concentration-response relationship was obtained comparing

the effect of ␣-DTx with its control. The data were ﬁtted with the

function Y = A2 + (A1–A2)/(1 + (x/IC50) ), where Y is the pharma-

cological effect of the substance under study, x is the substance

concentration, A1 and A2 are the maximum and the minimum

effects, IC50 is the concentration at which 50% of the effect is

obtained and p is the Hill slope constant. To deﬁne the statisti-

cal signiﬁcance a paired Student’s t-test was used and P ≤ 0.05 was

considered as signiﬁcant. Experimental data are presented as the

mean ± standard error.

2.5. Structural analyses

Electrostatic calculations were performed over crystal (␣-DTx,

PDB code: 1dtx, chain A) or NMR (PcTx1, PDB code: 2KNI; APETx2,

PDB code: 1WXN) structures with PDB2PQR [16] using default

parameters and PROPKA [17] to assign protonation states (at pH

6.1 or 7.4) and APBS [18]. Results were visualized with VMD [19],

which allows computing and depicting the dipole moment over

PQR ﬁles, i.e., PDB ﬁles where B-factor columns are replaced by

per-atom charge and radius.

3. Results

Fig. 1. Effect of ␣-DTx on ASIC currents. (A) Concentration-response curves of ␣-DTX

A total of 64 DRG neurons were successfully recorded. The mean inhibitory action on the ASIC currents of DRG neurons, in the sustained application

membrane capacitance of the cells was 40 ± 1.7 pF, corresponding (n = 41, circles, toxin was applied 20 s before the acid pulse and during the 5 s acid

pulse) and in co-application (n = 23, triangles, toxin was applied during the 5 s acid

to an estimated neuron diameter of 44 ± 5 ␮m. No signiﬁcant corre-

pulse). (B) Representative traces showing the ASIC current under control condition,

lation between membrane capacitance and any of the parameters

after sustained application of ␣-DTX and after washout (2 min). Dotted lines indicate

measured in ASIC currents (I , I /I ) was found. The

peak des, end peak the zero current, black bars the duration of the acid pulse and gray bars that of ␣-

current activated by the acidic solution varied from a fast transient DTX application. (C) Experiment showing that the co-application of ␣-DTX with the

acid pulse had no signiﬁcant effect (P > 0.05) on the ASIC current.

current with little or no steady-state component, to a current with

a small sustained component at the end of the acid pulse, indicating

that diverse ASICs participate in the generation of the macroscopic

minutes, in contrast with the effect on the peak of the inward ASIC

current [14,15]. For the 64 neurons, the control Ipeak was 5 0.6 nA,

current which was reversible after one minute washout of the toxin.

± ±

Iend = 96 11 pA, des = 410 36 ms.

The sustained application of ␣-DTx (prior to and during the

acid pulse) reduced the peak amplitude of the ASIC currents in 4. Discussion

a concentration-dependent manner over the assayed dose range

␣

␮ ␣

(Fig. 1A and B). In the presence of 3 M -DTx the Ipeak amplitude -DTx was found to reversibly inhibit the peak of ASIC currents

was reduced by 94 ± 1.2 %, from 8.9 ± 1.6 nA to 0.5 ± 0.07 nA (n = 5; from rat DRG neurons in a concentration-dependent manner with-

≤ ± ±

p 0.05), the Iend was also reduced by 92 3.6%, from 120 10 pA out signiﬁcantly affecting the time course of desensitization and

to 10 ± 3 pA (p ≤ 0.05), and no signiﬁcant change was found on the with no effect on the sustained component except for the con-

␮ ␣

des. The analysis of the concentration-response relationship for the centration of 3 M at which -DTx also inhibited the sustained

± ␮

Ipeak inhibition yielded an IC50 of 0.8 0.17 M and a Hill slope of component of the current. This effect is comparable to those of the

1.9 0.4. For the Iend no concentration response dependence was anemone peptide PhcrTx1 [20] and the ASIC blocker amiloride [21].

found and concentrations of ␣-DTX lower than 3 ␮M produced no In our experimental conditions, no effect was observed when ␣-

signiﬁcant effects. The effect of ␣-DTX was fully reversible after DTx was co-applied with the pH drop, indicating that the inhibitory

washing for 1 or 2 min, except at 3 ␮M, concentration at which action requires that the channel is in the closed state. Alternatively

the recovery took about 5 min. When ␣-DTx was co-applied (0.1, this lack of inhibition could reﬂect a slow onset of the interaction

0.3, 1 and 3 ␮M) with the acid pulse (n = 12, 5, 6 and 2, respec- with ASIC channels.

tively), no signiﬁcant effect was observed on any of the ASIC current In some experiments a transient outward current was evident

parameters (Fig. 1A–C). immediately after the end of the acid pulse. This current component

+ +

In some experiments (n = 14), a transient outward current com- could be due to the Na -dependent K conductance (KNa) which is

+ −

ponent appeared immediately after the end of the acid pulse (Fig. 2). activated by intracellular Na and Cl . KNa channels are homo or

Both the sustained application (n = 8, Fig. 2A) and the co-application heterotetramers of two subunits encoded by the Slick (Slo2.1) and

(n = 6, Fig. 2B) of ␣-DTx abolished this current at the concentra- Slack (Slo2.2) genes and are known to be expressed in DRG neurons

tions tested (30 nM and 100 nM for sustained application; 100 nM, [22,23]. Under control conditions KNa channels would be triggered

300 nM and 1 ␮M for co-application). The effect of ␣-DTx in the by the sodium inﬂux via ASICs. The fact that the sustained applica-

outward current was reversible only after washing for about ﬁve tion of ␣-DTx abolished the outward current component could be

A. Báez et al. / Neuroscience Letters 606 (2015) 42–47 45

Fig. 2. ␣-DTx inhibited the outward current component after the end of the acid pulse. (A) The sustained application of ␣-DTX produced a signiﬁcant inhibition of both the

inward (ASIC) current and the outward component at the end of the acidic pulse. Note that ASIC current is completely recovered after washing while the outward component

remained blocked. (B) The co-application of ␣-DTX did not produced an inhibition of the inward (ASIC) current but the outward current is clearly reduced. Dotted lines

indicate the zero current, black bars the duration of the acid pulse and gray bars that of ␣-DTX application.

due to a decrease in sodium inﬂux through ASICs [24]. However, Cys5–Cys55 bond. Key residues in the interaction appear to be a

because the co-application of the toxin with the acid pulse also protruding Lys and a close hydrophobic amino acid; whereas an

inhibited the outward peak current, it is not possible to rule out a enrichment of basic side chains at sites forming an interface with

direct action of ␣-DTx on the KNa channels, or the participation of the channel has also been a consistent ﬁnding [26]. Other com-

other channels or transport systems being activated by the acidic pounds with known inhibitory action on ASICs are characterized by

pulse. a strong positive charge at physiological pH. For example, PhcrTx1

Dendrotoxins are basic proteins with a net positive charge at has a net charge of around +5.0 at pH 7.4 [20], and a similar charge

neutral pH; Arg and Lys residues conforming a cationic domain has been observed for compounds such as aminoglycosides [15]

could, in principle, bind to anionic regions in the pore of potas- and FMRFamide-related peptides [27]. A cationic domain on the

sium channels [25]. When analyzed in more detail, the potassium surface of ␣-DTx may play an important role in the interaction

channel-blocking site of ␣-DTx and related toxins is formed by of the toxin with some functional domains of the ASIC channels,

residues from the N-terminus and the beta-turn region of the such as those related with their sensitivity to protons or Ca . Fur-

Kunitz domain, brought close to each other by the conserved thermore, the charge anisotropy described for ASIC inhibitors as

Fig. 3. Structural and electrostatic comparison of ␣-DTx (1DTX) with other ASIC inhibitors. Spatial location of solvent-exposed residues conforming the basic/aromatic and

basic/hydroxyl clusters in PcTx1 and APETx2 (as proposed by Chagot et al. [28]) and ␣-DTx. The relative dipole moments for the molecules are shown. The relative dipole

moments for the molecules are shown. Labeled residues for ␣-DTx are both solvent exposed and conserved in other dendrotoxins, including DTx-K and ı-DTx. The color code

is green for polar uncharged, blue for basic, red for acidic and purple for aromatic residues. PDB accession codes are indicated between parentheses for each protein. Below

are the values for dipole moments at pH 6.1 and 7.4, which point to a higher pH-sensitivity for ␣-DTx.

46 A. Báez et al. / Neuroscience Letters 606 (2015) 42–47

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