Role of the outer pore domain in transient receptor potential vanilloid 1 dynamic permeability to large cations.

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.

Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C.

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-associated mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Many cold sensitive peripheral neurons of the mouse do not express TRPM8 or TRPA1.

Neurons of the peripheral nervous system detect changes in temperature through activation of specialised ion channels. Members of the transient receptor potential family TRPM8 and TRPA1 are candidates for the principal transducers of cold stimuli. Using ratiometric calcium imaging we now show that 19% of acutely dissociated mouse dorsal root (DRG) and 45% of superior cervical ganglia (SCG) neurons responded to a brief cold stimulus. Amongst cold-responsive DRG neurons 34+/-2% responded to the TRPM8 agonist menthol, 18+/-3% to the TRPA1 agonist mustard oil and 5% to both stimuli. A third of the cold-sensitive neurons did not respond to any TRP channel agonist. Cold-sensitive neurons of the SCG did not respond to menthol and only 3% responded to mustard oil. The threshold of SCG neurons was at significantly cooler temperatures than that of DRG neurons. Using real-time PCR, TRPA1 was expressed over 100-fold more in DRG than SCG, while TRPM8 was present in DRG only. The relatively small amount of TRPA1 transcript present in SCG did not correlate with the high level of cold sensitivity. We conclude that cold sensitivity in sympathetic neurons and in a significant proportion of sensory neurons is generated in the absence of TRPM8 and TRPA1.