The vanilloid receptor [transient receptor potential (TRP)V1, also known as VR1] is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible contributions of terminal regions to vanilloid receptor function remain elusive. In the present study, C-terminal truncations of rat TRPV1 have been constructed to characterize the contribution of the cytoplasmic C-terminal region to TRPV1 function and to delineate the minimum amount of C tail necessary to form a functional channel. The truncation of 31 residues was sufficient to induce changes in functional properties of TRPV1 channel. More pronounced effects of C-terminal truncation were seen in mutants lacking the final 72 aa. These changes were characterized by a decline of capsaicin-, pH-, and heat-sensitivity; progressive reduction of the activation thermal threshold (from 41.5 to 28.6 degrees C); and slowing of the activation rate of heat-evoked membrane currents (Q10 from 25.6 to 4.7). The voltage-induced currents of the truncated mutants exhibited a slower onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1 C-terminal domain (155 residues) resulted in a nonfunctional channel. These results indicate that the cytoplasmic COOH-terminal domain strongly influences the TRPV1 channel activity, and that the distal half of this structural domain confers specific thermal sensitivity.

• MeSH
• Cell Line MeSH
• Electric Conductivity MeSH
• Immunohistochemistry MeSH
• Capsaicin pharmacology   MeSH
• Hydrogen-Ion Concentration MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Mutation MeSH
• Protein Kinase C metabolism   MeSH
• Protons MeSH
• Receptors, Drug chemistry   genetics   physiology   MeSH
• Amino Acid Sequence MeSH
• Sequence Deletion MeSH
• Sequence Alignment MeSH
• Serine genetics   MeSH
• Protein Structure, Tertiary MeSH
• Hot Temperature MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Capsaicin MeSH
• Protein Kinase C MeSH
• Protons MeSH
• Receptors, Drug MeSH
• Serine MeSH

1. The effects of capsaicin, acidic pH, ATP, kainate and GABA on currents generated by noxious heat were studied in cultured dorsal root ganglion (DRG) neurones (< 20 microm in diameter) isolated from neonatal rats. The patch clamp technique was used to record membrane currents or changes of membrane potential. 2. In agreement with previous results, inward membrane currents (I(heat)) induced by a 3 s ramp of increasing temperature from room temperature (approximately 23 degrees C) to over 42 degrees C varied greatly between cells (-100 pA to -2.4 nA at 48 degrees C) and had a temperature coefficient (Q(10)) > 10 over the range of 43-52 degrees C. 3. Capsaicin potentiated the heat-induced current even when capsaicin, at room temperature, had little or no effect on its own. In cells in which capsaicin induced no or very small membrane current at room temperature (< 50 pA), I(heat) exhibited detectable activation above 40 degrees C and increased 5.1 +/- 1.1 (n = 37) and 6.3 +/- 2.0 (n = 18) times at 0.3 and 1 microM capsaicin, respectively. 4. A rapid decrease in extracellular pH from 7.3 to 6.8, 6.3 or 6.1 produced an inward current which inactivated in ~5 s either completely (pH 6.8 or 6.3) or leaving a small current (approximately 50 pA) for more than 2 min (pH 6.1). After inactivation of the initial low pH-induced current, I(heat) at 48 degrees C increased 2.3 +/- 0.4 times at pH 6.8, 4.0 +/- 0.6 times at pH 6.3 and 4.8 +/- 0.8 times at pH 6.1 with a Q(10) > 10 (n = 16). 5. ATP (n = 22), kainate (n = 7) and GABA (n = 8) at 100 microM, produced an inactivating inward current in all heat-sensitive DRG neurones tested. During inactivation and in the presence of the drug, I(heat) was increased slightly with ATP and unaffected with kainate and GABA. These agents apparently do not directly affect the noxious heat receptor. 6. The results indicate a novel class of capsaicin-sensitive cells, in which capsaicin evokes no or very small inward current but nevertheless increases sensitivity to noxious heat.

• MeSH
• Adenosine Triphosphate pharmacology   MeSH
• Electric Conductivity MeSH
• Extracellular Space metabolism   MeSH
• Capsaicin pharmacology   MeSH
• Hydrogen-Ion Concentration MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Kainic Acid pharmacology   MeSH
• Acids pharmacology   MeSH
• Patch-Clamp Techniques MeSH
• Neurons, Afferent drug effects   physiology   MeSH
• Nociceptors physiology   MeSH
• Ganglia, Spinal cytology   drug effects   physiology   MeSH
• Hot Temperature * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Capsaicin MeSH
• Kainic Acid MeSH
• Acids MeSH

1. Membrane currents induced by noxious heat (Iheat) were studied in cultured dorsal root ganglion (DRG) neurones from newborn rats using ramps of increasing temperature of superfusing solutions. 2. Iheat was observed in about 70 % of small (< 25 microm) DRG neurones. At -60 mV, Iheat exhibited a threshold at about 43 C and reached its maximum, sometimes exceeding 1 nA, at 52 C (716 +/- 121 pA; n = 39). 3. Iheat exhibited a strong temperature sensitivity (temperature coefficient over a 10 C temperature range (Q10) = 17.8 +/- 2.1, mean +/- s.d., in the range 47-51 C; n = 41), distinguishing it from the currents induced by capsaicin (1 microM), bradykinin (5 microM) and weak acid (pH 6.1 or 6.3), which exhibited Q10 values of 1.6-2.8 over the whole temperature range (23-52 C). Repeated heat ramps resulted in a decrease of the maximum Iheat and the current was evoked at lower temperatures. 4. A single ramp exceeding 57 C resulted in an irreversible change in Iheat. In a subsequent trial, maximum Iheat was decreased to less than 50 %, its threshold was lowered to a temperature just above that in the bath and its maximum Q10 was markedly lower (5.6 +/- 0.8; n = 8). 5. DRG neurones that exhibited Iheat were sensitive to capsaicin. However, four capsaicin-sensitive neurones out of 41 were insensitive to noxious heat. There was no correlation between the amplitude of capsaicin-induced responses and Iheat. 6. In the absence of extracellular Ca2+, Q10 for Iheat was lowered from 25.3 +/- 7.5 to 4. 2 +/- 0.4 (n = 7) in the range 41-50 C. The tachyphylaxis, however, was still observed. 7. A high Q10 of Iheat suggests a profound, rapid and reversible change in a protein structure in the plasma membrane of heat-sensitive nociceptors. It is hypothesized that this protein complex possesses a high net free energy of stabilization (possibly due to ionic bonds) and undergoes disassembly when exposed to noxious heat. The liberated components activate distinct cationic channels to generate Iheat. Their affinity to form the complex at low temperatures irreversibly decreases after one exposure to excessive heat.

• MeSH
• Bradykinin pharmacology   MeSH
• Ion Channels metabolism   MeSH
• Capsaicin pharmacology   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Membrane Potentials drug effects   MeSH
• Neurons, Afferent drug effects   metabolism   MeSH
• Receptors, Drug metabolism   MeSH
• Ganglia, Spinal cytology   drug effects   metabolism   MeSH
• Tachyphylaxis physiology   MeSH
• Temperature MeSH
• Calcium metabolism   MeSH
• Hot Temperature * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Bradykinin MeSH
• Ion Channels MeSH
• Capsaicin MeSH
• Receptors, Drug MeSH
• Calcium MeSH