Chris Bladen

Molecular characterization of a type II cyclic GMP-dependent protein kinase expressed in the rat brain

Abstract: We applied reverse transcription‐PCR to examine the gene expression of cyclic GMP (cGMP)‐dependent protein kinase in the rat brain. A PCR product with the size predicted from the type II cGMP‐dependent protein kinase (cGK II) cDNA was detected in various regions of the brain, with highest expression in the thalamus. The amplified product of this cDNA was subcloned, sequenced, and consequently shown to be cGK II. Northern analysis confirmed that this kinase was highly expressed in the thalamus. In situ hybridization with riboprobes derived from this cDNA indicated that cGK II mRNA was highly expressed in the outer layers of the cortex, the septum, amygdala, and olfactory bulb with highest levels in the thalamus. High amounts of cGK II mRNA were also found in specific brainstem loci, including the medial habenula, the subthalamic nucleus, the locus ceruleus, the pontine nucleus, the inferior olivary nuclei, and the nucleus of the solitary tract. Only low levels of cGK II mRNA were detected in the striatum, cerebellum, and hippocampus. These data suggest that the effects of guanylyl cyclase activators, such as nitric oxide and the atriopeptides, in various regions of the CNS may be mediated through cGK II.

Extended Spectrum of Idiopathic Generalized Epilepsies Associated with CACNA1H Functional Variants

The relationship between genetic variation in the T‐type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and determine the electrophysiological effects of these variants in relation to proposed mechanisms underlying seizures.

Effects of Cav3.2 channel mutations linked to idiopathic generalized epilepsy

Heron and colleagues (Ann Neurol 2004;55:595–596) identified three missense mutations in the Cav3.2 T‐type calcium channel gene (CACNA1H) in patients with idiopathic generalized epilepsy. None of the variants were associated with a specific epilepsy phenotype and were not found in patients with juvenile absence epilepsy or childhood absence epilepsy. Here, we introduced and functionally characterized these three mutations using transiently expressed human Cav3.2 channels. Two of the mutations exhibited functional changes that are consistent with increased channel function. Taken together, these findings along with previous reports, strongly implicate CACNA1H as a susceptibility gene in complex idiopathic generalized epilepsy. Ann Neurol 2005

Expression of the olfactory cyclic nucleotide gated channel (CNG1) in the rat brain

The expression of the olfactory cyclic nucleotide-gated channel (CNG1) was studied in the rat brain. Using RT-PCR, levels of CNG1 mRNA were determined relative to the expression of a constantly expressed gene, alpha-tubulin. RT-PCR showed that CNG1 mRNA was detectable in the pituitary gland, the olfactory bulb, and the cerebellum of adult and 5-day-old rats. A 3.4 kb mRNA was detected in the olfactory bulb by Northern blotting. In situ hybridization analysis showed that CNG1 mRNA expression is present in the olfactory bulb and in the Purkinje cells of the cerebellum. RT-PCR studies on Purkinje cell-enriched cultures obtained from the cerebellum of 16-day-old embryos (E16) confirmed the expression of CNG1 mRNA in these neurones. Our results show that CNG1 is not restricted to the olfactory epithelium but is also present in specific regions of the brain. These results suggest that cyclic nucleotides may act in the regions that possess CNG1 gene expression to affect the electrical activity of certain neurones directly.

A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

&NA; Voltage‐gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including NaV1.7 and NaV1.8 sodium channels and CaV3.2 T‐type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow‐inactivated state. Slow inactivation of voltage‐gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state‐dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot‐plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state‐dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics. A novel organic compound stabilizes slow‐inactivated sodium and calcium channels to reduce the excitability of nociceptors and dorsal horn neurons and attenuate neuropathic pain signaling.

Functional analysis of Cav3.2 T-type calcium channel mutations linked to childhood absence epilepsy

Summary:  Purpose: Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis.

Synthesis and evaluation of 1,4-dihydropyridine derivatives with calcium channel blocking activity.

Abstract1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors.

Common Mechanisms of Drug Interactions with Sodium and T-Type Calcium Channels

Voltage-gated sodium (Nav) and calcium (Cav) channels play important roles in physiological processes, including neuronal and cardiac pacemaker activity, vascular smooth muscle contraction, and nociception. They are thought to share a common ancestry, and, in particular, T-type calcium (T-type) channels share structural similarities with Nav channels, both with regard to membrane topology and with regard to gating kinetics, including rapid inactivation. We thus reasoned that certain drugs acting on Nav channels may also modulate the activities of T-type channels. Here we show that the specific Nav1.8 blocker 5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide (A803467) tonically blocks T-type channels in the low micromolar range. Similarly to Nav1.8, this compound causes a significant hyperpolarizing shift in the voltage dependence of inactivation and seems to promote a slow inactivation-like phenotype. We further hypothesized that the structural similarity between T-type and Nav channels may extend to structurally similar drug-binding sites. Sequence alignment revealed several highly conserved regions between T-type and Nav channels that corresponded to drug-binding sites known to alter voltage-dependent gating kinetics. Mutation of amino acid residues in this regions within human Cav3.2 T-type channels altered A803467 blocking affinity severalfold, suggesting that these sites may be exploited for the design of mixed T-type and Nav channel blockers that could potentially act synergistically to normalize aberrant neuronal activity.

Characterization of Novel Cannabinoid Based T‑Type Calcium Channel Blockers with Analgesic Effects

Low-voltage-activated (T-type) calcium channels are important regulators of the transmission of nociceptive information in the primary afferent pathway and finding ligands that modulate these channels is a key focus of the drug discovery field. Recently, we characterized a set of novel compounds with mixed cannabinoid receptor/T-type channel blocking activity and examined their analgesic effects in animal models of pain. Here, we have built on these previous findings and synthesized a new series of small organic compounds. We then screened them using whole-cell voltage clamp techniques to identify the most potent T-type calcium channel inhibitors. The two most potent blockers (compounds 9 and 10) were then characterized using radioligand binding assays to determine their affinity for CB1 and CB2 receptors. The structure–activity relationship and optimization studies have led to the discovery of a new T-type calcium channel blocker, compound 9. Compound 9 was efficacious in mediating analgesia in mouse models of acute inflammatory pain and in reducing tactile allodynia in the partial nerve ligation model. This compound was shown to be ineffective in Cav3.2 T-type calcium channel null mice at therapeutically relevant concentrations, and it caused no significant motor deficits in open field tests. Taken together, our data reveal a novel class of compounds whose physiological and therapeutic actions are mediated through block of Cav3.2 calcium channels.

Autoradiographic localization of [3H]-cyclic GMP binding sites in the rat brain

Both the atriopeptides and nitric oxide act in the nervous system by activating guanylyl cyclases to stimulate the production of cyclic GMP. Thus a key to understanding the roles of these messengers is to understand the functions of cyclic GMP in the nervous system. Three potential targets for cyclic GMP have been identified, phosphodiesterases, protein kinases and ion channels. In this study we describe a method using autoradiography to localize specific [3H]-cGMP binding sites in the brain. The specific binding of [3H]-cGMP to rat brain sections was saturable (Bmax = 1.5 pmol/mg protein) and of high affinity (KD = 164 nM). The pharmacological characteristics were consistent with binding to the cGMP-dependent protein kinase. Highest densities of binding were seen in the medial habenula, basal ganglia, locus ceruleus and nucleus of the solitary tract. The CA1 pyramidal cells of the hippocampus, the neocortex, thalamus and cerebellum were also labelled. This method should prove useful in studies of potential targets for cyclic GMP in the brain.