Edward B. Stevens

Ion Channels as Therapeutic Targets: A Drug Discovery Perspective

Ion channels are membrane proteins expressed in almost all living cells. The sequencing of the human genome has identified more than 400 putative ion channels, but only a fraction of these have been cloned and functionally tested. The widespread tissue distribution of ion channels, coupled with the plethora of physiological consequences of their opening and closing, makes ion-channel-targeted drug discovery highly compelling. However, despite some important drugs in clinical use today, as a class, ion channels remain underexploited in drug discovery and many existing drugs are poorly selective with significant toxicities or suboptimal efficacy. This Perspective seeks to review the ion channel family, its structural and functional features, and the diseases that are known to be modulated by members of the family. In particular, we will explore the structure and properties of known ligands and consider the future prospects for drug discovery in this challenging but high potential area.

The sodium channel β‐subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart

1 Cardiac sodium channels are composed of a pore‐forming α‐subunit, SCN5a, and one or more auxiliary β‐subunits. The aim of this study was to investigate the role of the recently discovered member of the β‐subunit family, SCN3b, in the heart. 2 Northern blot and Western blot studies show that SCN3b is highly expressed in the ventricles and Purkinje fibres but not in atrial tissue. This is in contrast to the uniform expression of SCN1b throughout the heart. 3 Co‐expression of SCN3b with the cardiac‐specific α‐subunit SCN5a in Xenopus oocytes resulted in a threefold increase in the level of functional sodium channel expression, similar to that observed when SCN1b was co‐expressed with SCN5a. These results suggest that both SCN1b and SCN3b improve the efficiency with which the mature channel is targeted to the plasma membrane. 4 When measured in cell‐attached oocyte macropatches, SCN3b caused a significant depolarising shift in the half‐voltage of steady‐state inactivation compared to SCN5a alone or SCN5a + SCN1b. The half‐voltage of steady‐state activation was not significantly different between SCN5a alone and SCN5a + SCN3b or SCN5a + SCN1b. 5 The rates of inactivation for SCN5a co‐expressed with either subunit were not significantly different from that for SCN5a alone. However, recovery from inactivation at −90 mV was significantly faster for SCN5a + SCN1b compared to SCN5a + SCN3b, and both were significantly faster than SCN5a alone. 6 Thus, SCN1b and SCN3b have distinctive effects on the kinetics of activation and inactivation, which, in combination with the different patterns of expression of SCN3b and SCN1b, could have important consequences for the integrated electrical activity of the intact heart.

β3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage‐gated sodium channel α‐subunits. However, of the auxiliary subunits, β1 is expressed preferentially in only large‐ and medium‐diameter neurons of the DRG while β2 is absent in all DRG cells. In view of this, we have compared the distribution of β1 in rat DRG and spinal cord with a novel, recently cloned β1‐like subunit, β3. In situ hybridization studies demonstrated high levels of β3 mRNA in small‐diameter c‐fibres, while β1 mRNA was virtually absent in these cell types but was expressed in 100% of large‐diameter neurons. In the spinal cord, β3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while β1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of β3 expression in DRG appears to correlate with the TTX‐resistant voltage‐gated sodium channel subunit PN3, we co‐expressed the two subunits in Xenopus oocytes. In this system, β3 caused a 5‐mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of β‐subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in β3 mRNA expression in small‐diameter sensory neurons of the ipsilateral DRG. These results show that β3 is the dominant auxiliary sodium channel subunit in small‐diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.

Developmental expression of the novel voltage-gated sodium channel auxiliary subunit β3, in rat CNS

1 We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Naβ1.1 and Naβ2.1 and the novel, recently cloned subunit, β3. 2 In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and β3 mRNA at embryonic stages whilst Naβ1.1 and Naβ2.1 mRNA was absent throughout this period. 3 Naβ1.1 and Naβ2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. β3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4 Emulsion‐dipped slides showed co‐localisation of β3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5 Co‐expression in Xenopus oocytes revealed that β3 could modify the properties of Nav1.3; β3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6 In conclusion, β3 is shown to be the predominant β subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage‐gated sodium channels during development.

Bombesin-like peptides depolarize rat hippocampal interneurones through interaction with subtype 2 bombesin receptors.

1 Whole‐cell patch‐clamp recordings were made from visually identified hippocampal interneurones in slices of rat brain tissue in vitro. Bath application of the bombesin‐like neuropeptides gastrin‐releasing peptide (GRP) or neuromedin B (NMB) produced a large membrane depolarization that was blocked by pre‐incubation with the subtype 2 bombesin (BB2) receptor antagonist [D‐Phe6,Des‐Met14]bombesin‐(6‐14)ethyl amide. 2 The inward current elicited by NMB or GRP was unaffected by K+ channel blockade with external Ba2+ or by replacement of potassium gluconate in the electrode solution with caesium acetate. 3 Replacement of external NaCl with Tris‐HCl significantly reduced the magnitude of the GRP‐induced current at ‐60 mV. In contrast, replacement of external NaCl with LiCl had no effect on the magnitude of this current. 4 Photorelease of caged GTPγS inside neurones irreversibly potentiated the GRP‐induced current at ‐60 mV. Similarly, bath application of the phospholipase C (PLC) inhibitor U‐73122 significantly reduced the size of the inward current induced by GRP. 5 Reverse transcription followed by the polymerase chain reaction using cytoplasm from single hippocampal interneurones demonstrated the expression of BB2 receptor mRNA together with glutamate decarboxylase (GAD67). 6 Although bath application of GRP or NMB had little or no effect on the resting membrane properties of CA1 pyramidal cells per se, these neuropeptides produced a dramatic increase in the number and amplitude of miniature inhibitory postsynaptic currents in these cells in a TTX‐sensitive manner.

Tissue distribution and functional expression of the human voltage-gated sodium channel β3 subunit

Abstract. This study investigated the distribution of β3 in human tissues and the functional effects of the human β3 subunit on the gating properties of brain and skeletal muscle α subunits. Using RT-PCR of human cDNA panels, β3 message was detected in brain, heart, kidney, lung, pancreas and skeletal muscle. Both αIIA and SkM1 expressed in Xenopus oocytes inactivated with a time course described by two exponential components representing fast and slow gating modes, while co-expression of human β3 with αIIA or SkM1 significantly increased the proportion of channels operating by the fast gating mode. In the presence of β3 a greater proportion of αIIA or SkM1 current was described by the fast time constant for both inactivation and recovery from inactivation. β3 caused a hyperpolarizing shift in the voltage dependence of inactivation of αIIA and reduced the slope factor. The voltage dependence of inactivation of SkM1 was described by a double Boltzmann equation. However, SkM1 co-expressed with β3 was described by a single Boltzmann equation similar to one of the Boltzmann components for SkM1 expressed alone, with a small positive shift in V1/2 value and reduced slope factor. This is the first study demonstrating that β3 is expressed in adult mammalian skeletal muscle and can functionally couple to the skeletal muscle α subunit, SkM1.

Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes

1 The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein‐gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild‐type subunits together with the m2 muscarinic receptor in Xenopus oocytes. 2 Co‐expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1–Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane. 3 Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine‐induced channel activity compared with the wild‐type Kir3.2 homomeric channel. Cell‐attached, single‐channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings. 4 Individually substituting the N‐ and C‐terminal tails of Kir3.1 for those of Kir3.2 showed that the C‐terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N‐terminal or core regions of this subunit. 5 The chimeric channel, 121, displayed a higher ratio of ACh‐induced to basal activity than the Kir3.1–Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for Gβγ. Our results suggest that substituting the Kir3.1 C‐terminal tail for the Kir3.2 tail promotes the opening conformational change of the Gβγ‐bound channel. 6 The core and C‐terminal regions of Kir3.1 independently conferred time dependence on voltage‐dependent activation. The time constant (τ) was between 5 and 10 ms and varied little over the voltage range −60 to −120 mV.

The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation

Background: Species-dependent pharmacology is an obstacle for TRPV1 antagonist development. Results: By exchanging the pore domains TRPV1 antagonist JYL-1421, which is modality-selective in rTRPV1 can be made modality-selective in hTRPV1 and vice-versa. Conclusion: The pore region is critical for the observed species differences. Significance: Thus, the findings are of significance for the development of more specific and selective TRPV1 antagonists. The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Discovery and Optimization of Selective Nav1.8 Modulator Series That Demonstrate Efficacy in Preclinical Models of Pain

Voltage-gated sodium channels, in particular Nav1.8, can be targeted for the treatment of neuropathic and inflammatory pain. Herein, we described the optimization of Nav1.8 modulator series to deliver subtype selective, state, and use-dependent chemical matter that is efficacious in preclinical models of neuropathic and inflammatory pain.

CHAPTER 4:Ion Channel Modulators

This chapter reviews the essential role of ion channels as drug targets in the sensory perception and conduction of pain. Beginning with the role of TRP channels as noxious environmental sensors, the chapter follows the creation of action potentials, their conduction through sodium and potassium channels through to the control of neurotransmitter release at the first synapse by voltage-gated calcium channels. This chapter discusses the therapeutic options that these mechanisms offer and the progress within the drug discovery industry to date.