Sigma Receptor Photolabeling and Sigma Receptor-mediated Modulation of Potassium Channels in Tumor Cells

Abstract

Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membranespanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and (؎)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (I K ). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition in I K , and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 M. SKF10047 also inhibited I K with a similar EC 50 (14 M). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5 -O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.Sigma receptors are widely distributed in neuronal and nonneuronal tissue and are distinguished by their ability to bind a broad range of chemically unrelated ligands, including (ϩ)-opiates, neuroleptic drugs, ditolylguanidine (DTG), 1 and phencyclidine-related compounds (1-3). Although the biological activity of ligands suggests that sigma receptors may be involved in behavioral, psychological, and motor functions (1-3), the cellular actions of sigma receptors are poorly understood. Recent studies in melanotrophs (4) and the neurohypophysis (5) showed that sigma receptor ligands inhibit voltage-activated potassium current (I K ), but the signal transduction pathways associated with sigma receptor activation remain unknown.Molecular characterization of sigma receptors has raised intriguing questions about how these receptors generate cellular responses. The high affinity sigma receptor photoprobe iodoazidococaine (IAC) has labeled a 26-kDa protein in rat liver, rat brain, and human placenta (6). Cloning studies have confirmed that both human and rodent sigma receptor cDNAs encode a 25.3-kDa protein (7-10). The protein ...

Sigma Receptors and Cancer

“…Modulation of K ϩ channels by pentazocine or SKF10047 persisted although nerve terminals were internally perfused with GTPfree solutions, the G-protein inhibitor GDP␤S, or the G-protein activator GTP␥S. In DMS-114 cells (a tumor cell line isolated from a small-cell lung carcinoma), perfusion with GDP␤S also failed to alter the response to SKF10047 (65). Similar negative results were obtained in tests for protein kinase involvement.…”

“…Internal perfusion of nerve terminals with the non-hydrolysable ATP analog AMPPcP had no effect on K ϩ current inhibition by 1 receptor drugs. Of particular significance was the observation that K ϩ channels present in excised outside-out patches were modulated by SKF10047 (64,65). This effect excluded a role for any soluble cytoplasmic factor.…”

“…Indeed, these authors went on to show that the 1 receptor forms an immunoprecipitating complex with ion channels both in rat neurohypophysis and when coexpressed in Xenopus oocytes (20). In summary, studies on 1 receptor modulation of K ϩ channels, to date, have deduced the signal transduction mechanism of 1 receptors (a) to be membrane delimited (64,65); (b) to be independent of G-protein coupling and protein phosporylation (63)(64)(65); (c) to be reconstitutible in a heterologous system (20); (d) not to require cytoplasmic factors (64); and (e) to necessitate the 1 receptor and the K ϩ channel to be in close proximity (64), probably to form a stable macro-molecular complex (20).…”

The Sigma1 Protein as a Target for the Non-genomic Effects of Neuro(active)steroids: Molecular, Physiological, and Behavioral Aspects

“…Indeed, from rat cortical synaptosomes, C6 glioma cells (74) or NCB-20 cells (127), DTG, (+)3-PPP, and haloperidol have been shown to facilitate hyperpolarization with a reversal potential corresponding to that of K + or to block tonic outward K + currents. In rat neurohypophysis, Wilke et al (128) have confirmed this notion by providing evidence that (+)pentazocine and (+)SKF-10,047 as well as DTG and haloperidol, but not DHEAS or progesterone, elicited a marked inhibition of K + currents. This latter study then suggested that σ drugs and steroids may act distinctly at the molecular level.…”

The Role of Sigma Receptors in Depression

“…+ currents An interaction between sigma receptors and K + channels was suggested by the observation that the sigma ligands DTG and (+)-pentazocine inhibited K + currents (50,53,54). Further investigations of this modulation suggested that a protein-protein interaction is the likely mechanism of signal transduction by sigma receptors as sigma ligands did not interact directly with K + channels (43,54).…”