Direct Inhibition of I_h by Analgesic Loperamide in Rat DRG Neurons

Abstract

. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I h ) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I h channels in rat DRG neurons. Loperamide blocked I h in a concentration-dependent manner, with an IC 50 ϭ 4.9 Ϯ 0.6 and 11.0 Ϯ 0.5 M for large-and small-diameter neurons, respectively. Loperamideinduced I h inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V 1/2 for I h steady-state activation. Loperamide block of I h was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 M loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I h . Inclusion of 10 M loperamide into the recording pipette did not affect I h voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I h inhibition. The observed loperamideinduced I h inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I h inhibition. Therefore we suggest that loperamide inhibits I h by direct binding to the extracellular region of the channel. Because I h channels are involved in pain processing, loperamide-induced inhibition of I h channels could provide an additional molecular mechanism for its analgesic action.

Hyperpolarization-Activated Cation Channels: From Genes to Function

“…The number of potential chemical entities related to I h block is increased by agents that have been shown to block I h in addition to their wellknown primary receptor. For example, it was recently found that loperamide, a potent -opioid-receptor agonist, blocks I h of DRGs with quite high affinity (IC 50 between 5 and 10 M depending on the DRG type) (404). In addition, capsazepine, a well-known inhibitor of the vanilloid-receptor (TRPV1), blocks HCN1 in a concentration-dependent manner (IC 50 ϭ 8 M) (154).…”

A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential–Sensitive Dye and FLIPR

“…10,22 Indeed, the estimated resting membrane potential of HCN1 channelexpressing HEK293H cells on FLIPR was -40 to -45 mV (see Fig. 1), which was significantly different from the test voltage step used in IonWorks recordings (-110 mV; see Methods).…”

“…8), whereas randomly selected ICL compounds that were inactive in the MPSD assay did not show any activity on HCN1 channels by IonWorks recordings (data not shown). In addition, we have recently reported a novel pharmacology for loperamide on native HCN channels in DRG neurons, 10 but our observation of the loperamide effect was first made using the HCN blocker MPSD assay followed by confirmation of loperamide functional activity on HCN recombinant channels by IonWorks recordings. 22 Furthermore, the functional activity of selected HCN blockers obtained from ICL screening was confirmed by ex-vivo assay of low magnesium-induced hyperexcitability in rat hippocampal slices.…”

“…9 In addition, involvement of HCN channels in regulating neuronal excitability, sensory processing, and the pathophysiology of pain has been extensively investigated in preclinical studies. 4,5,10,11 HCN channels were shown to drive the frequency of ectopic discharges in A δ and C-fibers that are commonly associated with mechanical allodynia in rat models of neuropathic and postoperative pain. Peripheral administration of the nonselective HCN channel blocker ZD7288 significantly attenuated mechanical allodynia induced by partial sciatic nerve injury and hind-paw incision.…”

Ο Ρόλος Των Οπιοειδών Στην Ηλεκτρική Αντίσταση Υπεζωκότα Και Περικαρδίου Προβάτου

“…Το φαινόμενο αυτό έχει συσχετισθεί με την εμφάνιση ανοχής και εξάρτησης από τη μορφίνη (190,193,195 Μ, προκάλεσε ισχυρή αναστολή της καλμοδουλίνης (209)(210)(211). Μία άλλη δράση της λοπεραμίδης σε συγκεντρώσεις της τάξης των 10 -6 Μ είναι η αναστολή των εξαρτώμενων από το δυναμικό διαύλων Ca 2+ τύπου L (208,212,239). Σε καλλιέργειες μεσοθηλιακών κυττάρων υπεζωκότα και περικαρδίου ποντικού, φάνηκε ότι οι L δίαυλοι Ca 2+ σε συνεργασία με άλλους μηχανισμούς συμβάλλουν στη διατήρηση της ενδοκυττάριας συγκέντρωσης Ca 2+ , που είναι εξαιρετικά σημαντική σε μεταβολικά ενεργά κύτταρα (229).…”