T-type Ca2+ channels are present in cardiovascular, neuronal and endocrine systems and are now receiving attention as novel therapeutic targets. Many drugs and compounds non-specifically block T-type Ca2+ channels. The rapid entry of calcium into cells through activation of voltage-gated calcium channels directly affects membrane potential and contributes to electrical excitability, repetitive firing patterns, excitation-contraction coupling, and gene expression. At pre synaptic nerve terminals, calcium entry is the initial trigger mediating the release of neurotransmitters via the calcium-dependent fusion of synaptic vesicles and involves interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex of synaptic release proteins. Physiological factors or drugs that affect either pre-synaptic calcium channel activity or the efficacy of calcium-dependent vesicle fusion have dramatic consequences on synaptic transmission, including that mediating pain signalling. The N-type calcium channel exhibits a number of characteristics that make it an attractive target for therapeutic intervention in chronic and neuropathic pain conditions. Certain dihydropyridine compounds, such as Efonidipine blocks activity on both L-type and T-type Ca2+ channels, which possibly underlies their excellent clinical profiles such as minimum reflex tachycardia and renal protection. Selective inhibitors of T-type Ca2+ channels, are powerful pharmacological tools for further studies and may lead to the development of novel therapeutic strategies.
Native calcium channels have been classified by both their electro physiological and pharmacological properties and are generally divided into low-threshold (T-types) and high threshold (L-, N-, P/Q- and R-types). The L-, N-, P/Q- and R-type channels typically activate at membrane potentials near -30 mV and display diverse kinetic, voltage-dependent and pharmacological properties. The availability of specific pharmacological agents targeting the high threshold channels has permitted elucidation of many of their physiological functions. The T-type calcium channels describe a class of molecules that transiently activate at relatively negative potentials (~ — 60 mV) and for which a general lack of high-affinity selective blockers has made their exact physiological contributions lag behind those of the high-voltage activated isoforms.
Immunohistochemical studies show that most neurons express multiple types of alpha and beta-subunits, although differences in the relative levels of expression of individual subtypes are found throughout the CNS. For example, the a1C and a1D L-types and aE R-type subunits are primarily localized on cell bodies and proximal dendrites, whereas the a1A P/Q-type and a1B N-type subunits are more highly distributed along the lengths of dendrites and at presynaptic terminals. These distinctions are not absolute because aE R-type channels are also detected in cerebellar Purkinje cell dendritic branches and a1A P/Q-type channels are abundantly expressed on Purkinje cell bodies. That each of the classes of calcium channels shows a distinct expression pattern likely reflects their specialized physiological roles. For example, that a1A (Cav2.1) and a1B (Cav2.2) subunits are concentrated at a large number of presynaptic terminals is consistent with a prominent role for N-type and P/Q-type channels in triggering neurotransmitter release. That the al (Cav1.2) and a1 (Cav1.3) L-type channels appear to be largely restricted to cell bodies and proximal dendrites suggests that they likely serve to increase calcium influx at the base of major dendrites in response to the summation of synaptic inputs from dendritic trees and to also regulate somatic calcium-dependent signalling pathways (e.g., gene regulation).
In addition to their normal physiological functions, calcium channels are also implicated in a number of human pathophysiological conditions including congenital migraine, cerebellar ataxia, neuropathic/chronic pain, angina, epilepsy, hypertension, ischemia, and some arrhythmias. The clinical treatment of some of these disorders has been aided by the development of therapeutic calcium channel antagonists that selectively target those L-type channels largely localized to smooth muscle and the heart.
N-type channels
Biochemical purification of native N-type channels using cone snail peptide toxins as affinity ligands show the channel is a hetero-oligomeric complex consisting of a1B (Cav2.2), b-, and a2d-subunits. There is no firm evidence that the N-type channel complex contains a•g-subunit similar to that found in the skeletal muscle L-type channel complex. The large a1B-subunit (—2300 amino acid; —260 kDa) contains the channel pore, selectivity filter, and voltage-sensing machinery and is the target of all known pharmacological agents. Exogenous expression studies show that the voltage-dependent and kinetic properties of the a1B N-type channel can be differentially affected by co-expression with the distinct b-subunit isoforms. The b-subunits also play a key role in N-type channel modulation via both G protein and protein kinase C-dependent mechanisms.
Whereas pharmacologically defined L-type and T-type currents are each encoded by multiple a subunit genes, the a1B-subunit is encoded by a single gene. The degree of similarity between N-type channels is significantly greater across species (e.g., rat a1B N-type vs human a1B N-type —91% identity) than within the same species between N-type channels and the next closely related P/Q-type and R-type channels (rat N-type vs rat a1A P/Q-type-61% identity; rat N-type vs rat aE R-type-55% identity). The higher degree of N-type channel similarity between species is consistent with the significant conservation of both biophysical and pharmacological properties between rat, rabbit, and human N-type channels.
RNA analyses show that a1B transcripts are exclusively expressed in neurons and neuroendocrine cells such as chromaffin cells. In the CNS, a1B N-type channel mRNA is found in the cerebral cortex, hippocampus, forebrain, midbrain, cerebellum, brainstem, and spinal cord. Alternatively spliced variants have been identified that differ in primary sequence in domain IIIS3-S4, IVS3-S4, II-III linker, and the carboxyl tail region. In some instances, these variants alter channel biophysical properties and exhibit cell type-specific expression patterns (e.g., brain vs peripheral neurons), and there is some suggestion that primary afferents may exclusively express a particular N-type variant. Although there have been no reports that specific alternatively spliced N-type channel variants alters pharmacological properties, splicing variants in homologous extracellular regions of the P/Q-type channel have been shown to affect the binding of certain peptide toxins.
At the subcellular level, immunostaining shows a1B-subunits to be predominantly but unevenly clustered along dendritic regions and more diffusely and less frequently on cell bodies throughout the CNS. These results are largely in agreement with autoradiographic analyses using radio labelled conotoxin-GVIA, although they suggest a more specific distribution compared with that reported for a monoclonal antibody against conotoxin-GVIA itself. In agreement with pharmacological analyses showing that blockade of N-type channels disrupts a portion of central neurotransmission, a1B-subunits are found highly concentrated at a limited subset of presynaptic terminals in the central and peripheral nervous systems.
N-type channel activity can be modulated by activation of a number of G protein-coupled receptors (GPCRs). Of particular relevance, N-type currents are inhibited by GPCRs implicated in nociception including opioid, cannabinoid, neuropeptide Y, and substance P. In the spinal cord, opioids likely produce their antinociceptive actions act via a combination of two major mechanisms, both of which act via G protein-dependent pathways. In the one instance, activation of opioid receptors (both pre- and postsynaptic) releases Gbg from the trimeric G-abg complex and Gbg then physically interacts with potassium channels to up-regulate channel activity, hyperpolarizing the cell and decreasing synaptic excitability. In the second instance, the same opioid receptor G protein-dependent pathway results in Gbg molecules directly binding to presynaptic N-type calcium channels (in 1:1 stoichiometry) stabilizing the closed state and resulting in a 10-fold increase in the first latency of channel opening. A direct consequence of decreasing presynaptic N-type channel activity is likely to significantly attenuate neurotransmitter release in response to subsequent incoming action potentials.
Evidence suggests that the opioid receptor-mediated regulation of N-type currents by Gbg is, however, significantly more complicated. For example, the inhibition by Gbg is strongly voltage dependent, can be relieved by rapid trains of action potentials, is affected by the nature of the calcium channel b-subunit associated with the N-type channel complex (e.g., b1 vs b2a), and the Gbg interaction can be itself inhibited by the PKC-dependent phosphorylation of the N-type channel . It is also possible that alternative splicing of exon might regulate the association of N-type Ca2+ channels with neuronal binding proteins that are involved in the pain pathway. For example, a recent report by Beedle et al. shows that the proximal C-terminal region of rat Cav2.2 Ca2C channels interact physically with opioid-likenociceptin/orphanin FQ peptide (NOP) receptors. NOP receptors have low constitutive activity and, therefore, Ca2+ channels complexed with NOP are inhibited tonically by G-protein bg subunits, even in the absence of receptor agonists (Figure 2). Consequently, upregulation of NOP receptor expression, which is observed following chronic treatment with morphine, results in increased, tonic G-protein-mediated inhibition of N-type channels. This, in turn, should prevent further inhibition by mu opioid peptide receptors and, potentially, contribute to morphine tolerance. In the context of the findings of Bell and colleagues, it is of interest to determine whether alternative splicing of exon of the gene encoding N-type Ca2+ channels regulates their association with NOP.
N-type channels as primary therapeutic target for pain intervention
To a large degree, the case for involvement of N-type channels in various pathophysiological conditions rests with studies using high-affinity, selective peptides targeting N-type channels, as well as more recent work examining mice in which the N-type channel has been genetically deleted.
Two cone snail peptides, w-conotoxin-GVIA and w-conotoxin-MVIIA (also called SNX-111, Ziconotide and Prialt), have been the workhorses of numerous biochemical, pharmacological, and physiological studies examining the properties and roles of N-type channels. The 27-amino acid w-conotoxin-GVIA peptide isolated from Conus geographus is a potent, selective, and irreversible inhibitor of N-type channels. Similarly, w-conotoxin-MVIIA, a 25-amino acid peptide from the venom of Conus magnus is a potent blocker of N-type channels although acts in a reversible manner. Due to their peptidic nature, the cone snail toxins are not orally available and they must be delivered directly into the CNS via intrathecal administration.
N-type channels are highly concentrated in both dorsal root ganglia cell bodies and also in the synaptic terminals they make in dorsal horn of the spinal cord (laminae I and II). These primary afferents (mainly C-fibers and A-S fibers) are implicated in the sensation of a variety of noxious painful stimuli including thermal, mechanical, and inflammatory.
T-type Ca2+ channels
T-type Ca2+ channel has properties different from those of the L-type such as more negative voltage range of activation and inactivation, rapid gating kinetics, and resistance to standard Ca2+ blockers . Three different cDNAs for T-type Ca2+ channels have been cloned, and information on their distribution and function is now accumulating. T-type Ca2+ channels are expressed throughout the body, including nervous tissue, heart, kidney, smooth muscle, and many endocrine organs. T-type Ca2+ channels in the brain are considered to be involved in repetitive low threshold firing and nociception. In the heart, T-type Ca2+ channels are expressed in the sinoatrial node and are considered to participate in cardiac pacemaking, but not in normal myocardial contraction. The expression of T-type Ca2+ channels was shown to be increased in immature hearts and proposed to be involved in the development of pathological conditions such as ventricular hypertrophy, myocardial infarction, cardiacc myopathy, heart failure, and atrial fibrillation.. T-type Ca2+ channels are expressed in various arteries and veins with a distribution different from that of L-type Ca2+ channels and are considered to participate in regulation of micro- circulation. T-type Ca2+ channels are also expressed in the smooth muscle of bronchi, ileum, colon, bladder, and uterus. Involvement of T-type Ca2+ channels in the secretion of various hormones has been postulated including aldosterone, renin, atrial natriuretic peptide, and insulin. Thus, T-type Ca2+ channels are now anticipated to be novel therapeutic targets for the treatment of various cardiovascular disorders such as heart failure, arrhythmia, and hypertension and neuronal disorders such as epilepsy and pain. Inhibition of T-type Ca2+ channels may result in long-term organ protection through improvement of local microcirculation and reduction of adverse hormonal effects.
Diversity of drugs with T-type Ca2+ channel blocking activity
Ca2+ channel blockers, which block L-type Ca2+ channels, are classified into three major groups, phenylalkylamines (verapamil, etc.), benzotiazepines (diltiazem, etc.) and dihydropyridines (nifedipine etc.). These drugs in general do not inhibit T-type Ca2+ channels at therapeutic concentrations, except for certain dihydropyridine compounds that have dual blocking action on the L-type and T-type Ca2+ channels. Ca2+ blockers with low specificity such as flunarizine and cinnarizine are reported to have T-type Ca2+ channel-blocking activity, but its relation to their therapeutic potential is uncertain.
Mibefradil is a tetralol derivative that blocks T-type Ca2+ channels at concentrations lower than that to block L-type Ca2+ channels. It appeared to be a promising drug for the treatment of hypertension and angina pectoris. It reduced blood pressure and heart rate without suppressing myocardial contraction. Mibefradil also had beneficial effects in various animal models of heart failure and improved survival rate in a rat chronic myocardial infarction model; anti-arrhythmic effects through T-type Ca2+ channel blockade possibly accompanied this. Unfortunately, however, the drug had to be withdrawn from the market due to drug interaction at the cytochrome P-450 3A4 enzyme which was unrelated to T-type Ca2+ channel blockade. Some of the pharmacological profiles of mibefradil appear to be shared by dihydropyridine Ca2+ blockers with blocking action on both L-type and T-type Ca2+ channels . It was recently reported that the L-type Ca2+ channel blocking activity of mibefradil was due to production of an active metabolite by intracellular hydrolysis, and that a non-hydrolyzable analogue of mibefradil selectively inhibits the T-type Ca2+ channel.
There are many other compounds that have been reported to inhibit the T-type Ca2+ channel, but most of them also affects other ion channels and transporters, and no specific inhibitor of the channel has been established at present. These include antiepileptic drugs phenytoin and ethosuximide, anesthetics iso-flurane and pentobarbital, and drugs acting on the central nervous system such as haloperidol and pimozide. As T-type Ca2+ channels are reported to be involved in neuron excitation and hormone secretion, it is possible that the T-type Ca2+ channel-blocking effect of these drugs contribute to their therapeutic potential. Kurtoxin, a peptide isolated from a venomous scorpion, was reported to inhibit T-type Ca2+ channels, but the toxin was found to block other types of Ca2+ channels as well .
Conclusion
The transmission of pain signals at the spinal level is crucially dependent on voltage-gated Ca2+ channels in nociceptive neurons. Pharmacological and gene-knockout studies implicate N-type Ca2+ channels as key mediators of nociceptive signalling in dorsal root ganglion (DRG) neurons, and as potential targets for the development of analgesic drugs. Furthermore, nociceptor-specific alternative splicing of the gene encoding N-type Ca2+ channels might provide strategies for splice-isoform-specific drug targeting. More recently, T-type Ca2+ channels have been implicated in the processing of pain signals at both spinal and thalamic levels. However, although inhibition of T-type channel activity in DRG neurons mediates analgesia, gene knock-out of T-type channels in the CNS is reported to increase the perception of visceral pain. So, the implications of these findings can be employed to design novel therapeutic strategies and contrast the role of T-type channels with that of N-type channels in pain transmission and analgesia.
AN Nagappa is faculty Department of Pharma Mangement, Manipal College of Pharmaceutical Sciences, Manipal and Lajwinder Kumar is faculty, Birla Institute of Technology and Sciences, Pilani