Research ReportSigma-1 receptor alters the kinetics of Kv1.3 voltage gated potassium channels but not the sensitivity to receptor ligands
Highlights
► S1R accelerates Kv1.3 current inactivation but not ligand sensitivity. ► Kv1.3 transmembrane domains interact with S1R. ► Distinct protein interaction domains likely mediate S1R modulation of Kv1.3.
Introduction
Sigma1 receptors (Sigma1R) are mostly endoplasmic reticulum-resident two-transmembrane chaperone proteins that bind psychotropic drugs and also drugs such as ketamine, haloperidol, and intravenous anesthetics relevant to clinical medicine (Cobos et al., 2008, Su et al., 2010, Yamada et al., 2006). Co-expression of the Sigma1R has been reported to enhance the sensitivity of several voltage-gated ion channels including calcium, sodium, and potassium channels to Sigma1R ligands (Zhang and Cuevas, 2002, Johannessen et al., 2009, Wilke et al., 1999). A mechanistic study using the Xenopus oocyte model suggests that the Sigma1R also present in the plasma membrane serve a novel role as a ligand-dependent auxiliary channel subunit protein through direct association with the Kv1.4 and Kv1.5 voltage-gated potassium channel proteins, affecting the ion channel physiology only in the presence of a Sigma1R ligand (Aydar et al., 2002).
In addition to these effects of Sigma1R on ion channels physiology, Sigma1R ligands have been reported to show immuno-modulatory effects. Suppression of splenocyte proliferation and NK cell activity (Liu et al., 1995, Carr et al., 1992), inhibition of mitogen induced lymphocyte proliferative response (Casellas et al., 1994), IL-10 mediated suppression of antitumor immunity (Zhu et al., 2003), and enhancement of monocyte transmigration (Yao et al., 2010) have been ascribed to various Sigma 1R ligands including haloperidol and ketamine (Leykin et al., 1997, Rofael et al., 2003, Ohta et al., 2009). The precise mechanisms of these immuno-modulatory effects remain largely unresolved.
Kv1.3 is the predominant voltage-gated potassium channel expressed in T lymphocytes and this ion channel plays a key role in T cell activation. The functional significance of Kv1.3 in T lymphocyte activation derives from the fact that this potassium channel in the plasma membrane maintains cell membrane at a hyperpolarized negative potential. The negative membrane potential maintains the driving force for Ca2 + allowing sustained influx of extracellular Ca2 + during antigen presentation at the T cell immunological synapse. The sustained influx of extracellular Ca2 + in coordination with Ca2 + released from the intracellular stores triggers Ca2 +-calmodulin-dependent phosphatase calcineurin. The consequent activation of the transcription factor NFAT is thought to be the critical signaling responsible for T cell activation (Panyi et al., 2004). As such, blocking plasma membrane Kv1.3 by extracellularly applied toxins has been shown to inhibit T cell activation in an experimental autoimmune encephalomyelitis model of multiple sclerosis, rheumatoid arthritis and type-1 diabetes (Beeton et al., 2001, Beeton et al., 2006). Development of a selective extracellular blocker of plasma membrane Kv1.3 as a novel immunosuppressant is an active area of basic and translational research.
Sigma1R are also present in T cells (Ganapathy et al., 1999) but whether this receptor affects Kv1.3 physiology is not known. To gain a better understanding of the potential immuno-modulatory role of Sigma1R ligands acting through Kv1.3, we investigated the effects of Sigma1R co-expression on Kv1.3 physiology and pharmacology in Xenopus oocytes. Given the earlier report that Sigma1R alters the ligand inhibition of Kv1.4 and Kv1.5 voltage gated potassium channels functioning as a ligand-dependent auxiliary protein to the channels, we hypothesized that the Sigma1R will modulate the ligand sensitivity of the closely related Kv1.3 channel.
Section snippets
Results
A two-electrode voltage clamp of the injected oocytes demonstrated voltage-activated slowly inactivating outward-going currents consistent with currents flowing through the Kv1.3 channels (Spencer et al., 1997, Kuppar, 1998, DeCoursey et al., 1985). No qualitative difference in the current morphology was seen in oocytes injected with cRNA for HA-tagged Kv1.3 alone or in combination with the FLAG-tagged Sigma1R (Fig. 1A). Reversal potentials were determined by a tail protocol (Fig. 1 B) and
Discussion
The Sigma1R when expressed in Xenopus oocyte was present in the plasma membrane in the correct cellular compartment for potential interaction with Kv1.3 as reported previously (Aydar et al., 2002) and confirmed here by a Western blot of the isolated oocyte plasma membrane. Sigma1R co-expression with Kv1.3 accelerated the voltage-dependent inactivation of this ion channel at all voltages examined identical to a previously reported ligand-independent effect of Sigma1R on Kv1.4. In contrast,
Molecular biology
Full-length cDNA for human Sigma1R variant 1 and human Kv1.3 were obtained from OriGene (Rockville, MD). An epitope tag (3 × HA or 3 × FLAG) was placed on the C-terminal of the cDNA through a polymerase chain reaction (PCR)-engineered Mlu I restriction enzyme site that replaced the native stop codon. The PCR amplified segments of the cDNA were sequenced to confirm its fidelity. The resulting epitope-tagged proteins are identical to the original sequence except for a threonine–arginine dipeptide
Acknowledgments
We thank Dr. Arnold Ruoho (Department of Pharmacology, UW Madison, WI) for introducing the senior author to the Sigma1R area of research and for constructive discussions. This research was partly supported by NIH RO1GM086401 (JY) and funds from the University of Wisconsin Department of Anesthesiology.
References (32)
- et al.
The sigma receptor as a ligand-regulated auxiliary potassium channel subunit
Neuron
(2002) - et al.
Immunopharmacological profile of SR31747: in vitro and in vivo studies on humoral and cellular responses
J. Immunolol.
(1994) - et al.
Two adaptor proteins differentially modulate the phosphorylation and biophysics of Kv1.3 ion channel by Src kinase
J. Biol. Chem.
(2002) - et al.
Voltage-dependent ion channels in T-lymphocytes
J. Neuroimmunol.
(1985) - et al.
Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2 +) signaling and cell survival
Cell
(2007) - et al.
Contribution of the selectivity filter to inactivation in potassium channels
Biophy. J.
(1999) - et al.
Short and long-term immunosuppressive effects of clozapine and haloperidol
Immunopharmacol.
(1997) - et al.
Sigma-1 receptors modulate functional activity of rat splenocytes
J. Neuroimmunol.
(1995) - et al.
C-type Inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism
Biophys. J.
(1995) - et al.
Ion channels and lymphocyte activation
Immunol. Lett.
(2004)