Paul Shartzer Dietrich

Functional Analysis of a Voltage‐Gated Sodium Channel and Its Splice Variant from Rat Dorsal Root Ganglia

Abstract: Neurons of the dorsal root ganglia (DRG) express a diversity of voltage‐gated sodium channels. From rat DRG we have cloned and functionally expressed a tetrodotoxin‐sensitive sodium channel α subunit, NaCh6/Scn8a/rPN4, and a splice variant, rPN4a. Primary structure analysis shows NaCh6/Scn8a/rPN4 to be highly homologous (99%) to NaCh6 and most likely represents the same transcript. The splice variation in rPN4a is homologous in sequence and location to that of rat brain I. Tissue distribution analyzed by RT‐PCR showed NaCh6/Scn8a/rPN4 to be expressed at its highest levels in rat brain, at moderate levels in spinal cord, and at lower levels in DRG, nodose ganglia, and superior cervical ganglia and to be absent from sciatic nerve, heart, and skeletal muscle. In contrast, rPN4a shows no expression in brain and low‐level expression in spinal cord, whereas in DRG its expression is comparable to that of NaCh6/Scn8a/rPN4. Functional analysis of these channels expressed in Xenopus oocytes showed that NaCh6/Scn8a/rPN4 and rPN4a exhibited similar properties, with V1/2≅−100 mV for steady‐state inactivation and V1/2≅−40 mV for activation. rPN4a recovered from inactivation significantly faster than NaCh6/Scn8a/rPN4. NaCh6/Scn8a/rPN4 was inhibited by tetrodotoxin with an IC50≅ 1 nM. Coexpression of the β1 subunit accelerated inactivation kinetics, but the β2 subunit was without effect.

Facilitatory interplay in α1A and β2 adrenoceptor function reveals a non-Gq signaling mode: implications for diversification of intracellular signal transduction

Agonist occupied α1-adrenoceptors (α1-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress α1- and β-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in α1A- and β2-AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca2+ mobilization to monitor in real time the activation of both Gq and Gs pathways. We found that coactivation of α1A- and β2-AR by the nonselective agonist NE or via a combination of the highly selective α1A-AR agonist A61603 and the β-selective agonist isoproterenol led to increases in Ca2+ influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca2+ from intracellular stores. This effect became more evident upon examination of an α1A-AR variant exhibiting a partial defect in coupling to Gq, and we attribute it to potentiation of a non Gq-pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca2+ entry channel blocker. We also found that stimulation with A61603 of a second α1A-AR variant entirely unable to signal induced no Ca2+ unless β2-AR was concomitantly activated. These results may be accounted for by the presence of α1A/β2-AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting α- and β-adrenoceptors.

Pharmacological Comparison of a Recombinant CB1 Cannabinoid Receptor with Its Ga16 Fusion Product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB1 agonist-mediated signal transduction via an engineered Gaα16 system is different than that of the Gi/o coupling normally preferred by the CB1 receptor, we transfected the human recombinant CB1 receptor (hCB1) or a fusion protein comprising the hCB1 receptor and Gα16 (hCB1-Gα16) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB1-Gaα16 fusion protein was found to be similar to that for hCB1: HU 210 > CP 55,940 ≥ SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [35S]GTPγS binding or inhibited forskolin-stimulated cAMP, presumably by coupling to Gi/o, in cells expressing hCB1 but not hCB1-Gα16. However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCBq-Gaα16 but not hCB1. These data demonstrate that ligand affinities for the hCB1, receptor are not affected by fusion to the Gα16 subunit. Furthermore, there is essentially no difference in the function of the hCB1, receptor when coupled to Gi/o, or Gα16 .