Direct Inhibition ofIhby Analgesic Loperamide in Rat DRG Neurons

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Abstract
Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are responsible for the functional hyperpolarization-activated current ( Ih) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited Ihchannels in rat DRG neurons. Loperamide blocked Ihin a concentration-dependent manner, with an IC50= 4.9 ± 0.6 and 11.0 ± 0.5 μM for large- and small-diameter neurons, respectively. Loperamide-induced Ihinhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V1/2for Ihsteady-state activation. Loperamide block of Ihwas 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 μM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of Ih. Inclusion of 10 μM loperamide into the recording pipette did not affect Ihvoltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible Ihinhibition. The observed loperamide-induced Ihinhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse Ihinhibition. Therefore we suggest that loperamide inhibits Ihby direct binding to the extracellular region of the channel. Because Ihchannels are involved in pain processing, loperamide-induced inhibition of Ihchannels could provide an additional molecular mechanism for its analgesic action.