A novel α-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.


Rotarod test.
Motor coordination and balance were tested as described previously 1,2 .
Three days before the test, animals were trained at a speed of 10 rpm for 10 min once a day. Kuming mice (20 ± 2 g) were placed on an accelerating rotarod treadmill (YLS-4C, Shandong Academy of Medical Sciences, China). The peptides (16.6, 83.2 and 166.3 g/kg) or saline were administered intramuscularly (i.m.) to the mice (n = 8) in a volume of 20 l. After 120 min, the mice were placed on the rotating and the rod was accelerated from 0 to 30 rpm over a 5 min interval. The time during which the mice remained balanced on the rod was recorded (5 min as the maximum time). All methods and experimental protocols were approved and carried out in accordance with the guidelines and regulations of the Beijing Institutes for Biological Sciences Animal Research Advisory Committee and conformed to the European Community directives for the care and use of laboratory animals.
2, 8 and 24 h after the administration (i.v.) of Eu1.6 or saline, each mouse was put into an individual box and adapted to the environment for 2 min, the total moving distance and time in 6 min were then recorded.
On the day seven, the abstinence syndrome was precipitated by administering an i.p. injection of naloxone (5.0 mg/kg, 400 l) 6 h after administration of same doses of 3 peptides, morphine and saline. Each mouse was immediately placed in a square observation box (30 cm x 30 cm x 50 cm) and the number of jumps was recorded over a 15 min period.
Cardiac and respiratory function test. 24 Beagle dogs were randomly divided into four groups (3 female/3 male per group). Animals were administrated intravenously with Eu1.6 freeze-dry powder (Eu1.6 freeze-dry powder consisted of 2 % Eu1.6, 1% methionine and 97% mannitol) at 1, 10, and 50 mg/kg dissolved in saline (1 ml/kg) or the vehicle solution (1 ml/kg). Animals were placed on a warming pad maintaining at 37 ºC and anesthetized by 1-2% isoflurane inhalation. Surface ECG (QRS, PRQ, and QT) was continuously monitored by inserting subcutaneous needle electrodes in a limb lead II electrograph. Cardio-haemodynamic parameters (heart rate, aortic blood pressure, systolic blood pressure, and mean blood pressure) were measured as described previously 3 . QTc were calculated using the Fridercia's rate equation as QT/RR0.33.A respiratory belt was placed at the position of each animal to record respiratory rate. All parameters were measured using MP150 data acquisition and

Electrophysiological recording of TTX-S and TTX-R Na + currents in DRG
neurons. Whole-cell voltage clamp recordings of voltage-gated ionic currents were made in rat DRG neurons which were acutely dissociated from 30-day old Sprague-Dawley rats and maintained in short-term primary culture according to the method described by Xiao and Liang 6 . All methods and experimental protocols were approved and carried out in accordance with the guidelines and regulations of the Beijing Institutes for Biological Sciences Animal Research Advisory Committee. DRG neurons with large diameter (>35 µm) and those with relatively small diameter (<20 µm) were chosen for measuring TTX-S and TTX-R Na + currents, respectively. TTX (final concentration at 200 nM) was used to isolate TTX-R Na + currents from TTX-S Na + currents. Electrophysiological recording was performed as described previously 7 .