HCN1 channels as targets for anesthetic and nonanesthetic propofol analogs in the amelioration of mechanical and thermal hyperalgesia in a mouse model of neuropathic pain.

Chronic pain after peripheral nerve injury is associated with afferent hyperexcitability and upregulation of hyperpolarization-activated, cyclic nucleotide-regulated (HCN)-mediated IH pacemaker currents in sensory neurons. HCN channels thus constitute an attractive target for treating chronic pain. HCN channels are ubiquitously expressed; analgesics targeting HCN1-rich cells in the peripheral nervous system must spare the cardiac pacemaker current (carried mostly by HCN2 and HCN4) and the central nervous system (where all four isoforms are expressed). The alkylphenol general anesthetic propofol (2,6-di-iso-propylphenol) selectively inhibits HCN1 channels versus HCN2-HCN4 and exhibits a modest pharmacokinetic preference for the periphery. Consequently, we hypothesized that propofol, and congeners, should be antihyperalgesic. Alkyl-substituted propofol analogs have different rank-order potencies with respect to HCN1 inhibition, GABA(A) receptor (GABA(A)-R) potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tertbutylphenol (2,6- and 2,4-DTBP, respectively) are more potent HCN1 antagonists than propofol, whereas 2,6- and 2,4-di-sec-butylphenol (2,6- and 2,4-DSBP, respectively) are less potent. In contrast, DSBPs, but not DTBPs, enhance GABA(A)-R function and are general anesthetics. 2,6-DTBP retained propofol's selectivity for HCN1 over HCN2-HCN4. In a peripheral nerve ligation model of neuropathic pain, 2,6-DTBP and subhypnotic propofol are antihyperalgesic. The findings are consistent with these alkylphenols exerting analgesia via non-GABA(A)-R targets and suggest that antagonism of central HCN1 channels may be of limited importance to general anesthesia. Alkylphenols are hydrophobic, and thus potential modifiers of lipid bilayers, but their effects on HCN channels are due to direct drug-channel interactions because they have little bilayer-modifying effect at therapeutic concentrations. The alkylphenol antihyperalgesic target may be HCN1 channels in the damaged peripheral nervous system.

Dopamine modulation of Ih improves temporal fidelity of spike propagation in an unmyelinated axon.

We studied how conduction delays of action potentials in an unmyelinated axon depended on the history of activity and how this dependence was changed by the neuromodulator dopamine (DA). The pyloric dilator axons of the stomatogastric nervous system in the lobster, Homarus americanus, exhibited substantial activity-dependent hyperpolarization and changes in spike shape during repetitive activation. The conduction delays varied by several milliseconds per centimeter, and, during activation with realistic burst patterns or Poisson-like patterns, changes in delay occurred over multiple timescales. The mean delay increased, whereas the resting membrane potential hyperpolarized with a time constant of several minutes. Concomitantly with the mean delay, the variability of delay also increased. The variability of delay was not a linear or monotonic function of instantaneous spike frequency or spike shape parameters, and the relationship between these parameters changed with the increase in mean delay. Hyperpolarization was counteracted by a hyperpolarization-activated inward current (I(h)), and the magnitude of I(h) critically determined the temporal fidelity of spike propagation. Pharmacological block of I(h) increased the change in delay and the variability of delay, and increasing I(h) by application of DA diminished both. Consequently, the temporal fidelity of pattern propagation was substantially improved in DA. Standard measurements of changes in excitability or delay with paired stimuli or tonic stimulation failed to capture the dynamics of spike conduction. These results indicate that spike conduction can be extremely sensitive to the history of axonal activity and to the presence of neuromodulators, with potentially important consequences for temporal coding.

Effects of ZD7288 on firing pattern of thermosensitive neurons isolated from hypothalamus.

The role of the hyperpolarization-activated current (Ih) mediated by HCN channels in temperature sensing by the hypothalamus was addressed. In warm-sensitive neurons (WSNs), exposure to ZD7288, an inhibitor of Ih mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, decreased their action potential amplitudes and frequencies significantly. By contrast, ZD7288 had little or no effect on temperature-insensitive neurons (TINs). Exposure of WSNs to ZD7288 led to a significant increase in the duration of the inter-spike interval and a reduction of Ih irreversibly. These results suggest that ZD7288 have the contrasting effects on the firing patterns of WSNs versus TINs, which implies HCN channels play a central role in temperature sensing by hypothalamic neurons.

Tanshinone IIA selectively enhances hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel instantaneous current.

Tanshinone IIA, one of the main active components from the Chinese herb Danshen, is widely used to treat cardiovascular diseases in Asian countries, especially in China. To further elucidate its heart rate-reducing and anti-ischemic mechanisms, here we investigated the effects of tanshinone IIA on hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels expressed in Xenopus oocytes using two-electrode voltage clamp techniques. When applied to the extracellular solution, 100 microM tanshinone IIA caused a slowing of activation and deactivation and an increase of minimum open probabilities (from 0.06 +/- 0.01 to 0.29 +/- 0.03, P<0.05) in HCN2 channels without shifting the voltage dependence of channel activation. Tanshinone IIA potently enhanced the amplitude of voltage-independent current (instantaneous current) of HCN2 at -90 mV in a concentration-dependent manner with an EC(50) of 107 microM. Similar but 2.3-fold less sensitivity to tanshinone IIA was observed in the HCN1 subtype. More significant effect on HCN2 and MiRP1 co-expression was observed. In conclusion, tanshinone IIA changed HCN channel gating by selectively enhancing the instantaneous current (one population of HCN channels), which resulted in the corresponding increment of minimum open probabilities, slowing channel activation and deactivation processes with little effect on the voltage-dependent current (another population of HCN channels).

HCN channels as targets for drug discovery.

Hyperpolarization- and Cyclic Nucleotide-gated (HCN) channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. The HCN family consists of four members (HCN1-4). HCN channels represent the molecular correlates of I(h) (also known as 'funny' I(f) and 'queer' I(q)), a hyperpolarization-activated current best known for its role in controlling heart rate and in the regulation of neuronal resting membrane potential and excitability. A significant body of molecular and pharmacological evidence is now emerging to support a role for these channels in the function of sensory neurons and pain sensation, particularly pain associated with nerve or tissue injury. As such, HCN channels may represent valid targets for novel analgesic agents. This evidence will be reviewed in this article. We will then summarize our efforts to develop and validate methods for screening for novel HCN channel blockers.

Reciprocal modulation of I (h) and I (TASK) in thalamocortical relay neurons by halothane.

By combining electrophysiological, immunohistochemical, and computer modeling techniques, we examined the effects of halothane on the standing outward current (I (SO)) and the hyperpolarization-activated current (I (h)) in rat thalamocortical relay (TC) neurons of the dorsal lateral geniculate nucleus (dLGN). Hyperpolarizing voltage steps elicited an instantaneous current component (I (i)) followed by a slower time-dependent current that represented I (h). Halothane reduced I (h) by shifting the voltage dependency of activation toward more negative potentials and by reducing the maximal conductance. Moreover, halothane augmented I (i) and I (SO). During the blockade of I (h) through Cs+, the current-voltage relationship of the halothane-sensitive current closely resembled the properties of a current through members of the TWIK-related acid-sensitive K+ (TASK) channel family (I (TASK)). Computer simulations in a single-compartment TC neuron model demonstrated that the modulation of I (h) and I (TASK) is sufficient to explain the halothane-induced hyperpolarization of the membrane potential observed in current clamp recordings. Immunohistochemical staining revealed protein expression of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel proteins HCN1, HCN2, and HCN4. Together with the dual effect of halothane on I (h) properties, these results suggest that I (h) in TC neurons critically depends on HCN1/HCN2 heterodimers. It is concluded that the reciprocal modulation of I (h) and I (TASK) is an important mechanism of halothane action in the thalamus.