Inflammatory mediators not only activate "pain-"sensing neurons, the nociceptors, to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. We demonstrate for the first time at the cellular level that heat-activated ionic currents were potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation was prevented in the presence of the selective PKA inhibitor PKI(14-22), suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits only translocated to the cell periphery when activated. The translocation and the current potentiation were abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation was greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia.

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Voltage-gated Na(+) channels are the primary targets of local anesthetics (LAs). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form part of the LA binding site. LAs inhibit binding of the neurotoxin batrachotoxin (BTX). Parts of the BTX binding site are located in D1-S6 and D4-S6. The affinity of BTX-resistant Na(+) channels mutated in D1-S6 (mu1-N434K, mu1-N437K) toward several LAs is significantly decreased. We have studied how residue mu1-N434 influences LA binding. By using site-directed mutagenesis, we created mutations at mu1-N434 that vary the hydrophobicity, aromaticity, polarity, and charge and investigated their influence on state-dependent binding and stereoselectivity of bupivacaine. Wild-type and mutant channels were transiently expressed in human embryonic kidney 293t cells and investigated under whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers showed a higher potency in all mutant channels compared with wild-type channels. These changes were not well correlated with the physical properties of the substituted residues. Stereoselectivity was small and almost unchanged. In inactivated channels, the potency of bupivacaine was increased in mutations containing a quadrupole of an aromatic group (mu1-N434F, mu1-N434W, mu1-N434Y), a polar group (mu1-N434C), or a negative charge (mu1-N434D) and was decreased in a mutation containing a positive charge (mu1-N434K). In mutation mu1-N434R, containing the positively charged arginine, the potency of S(-)-bupivacaine was selectively decreased, resulting in a stereoselectivity (stereopotency ratio) of 3. Similar results were observed with cocaine but not with RAC 109 enantiomers. We propose that in inactivated channels, residue mu1-N434 interacts directly with the positively charged moiety of LAs and that D1-S6 and D4-S6 form a domain-interface site for binding of BTX and LAs in close proximity.