The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 °C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 °C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251-E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3.

• Klíčová slova
• ankyrin repeat, nociception, noxious heat, transient receptor potential, transient receptor potential vanilloid 1 (TRPV1),
• MeSH
• cytoplazma metabolismus   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPV chemie   genetika   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• simulace molekulární dynamiky MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• podjednotky proteinů MeSH
• TRPV3 protein, human MeSH Prohlížeč

The transient receptor potential ankyrin 1 (TRPA1) channel is a Ca(2+)-permeable cation channel whose activation results from a complex synergy between distinct activation sites, one of which is especially important for determining its sensitivity to chemical, voltage and cold stimuli. From the cytoplasmic side, TRPA1 is critically regulated by Ca(2+) ions, and this mechanism represents a self-modulating feedback loop that first augments and then inhibits the initial activation. We investigated the contribution of the cluster of acidic residues in the distal C terminus of TRPA1 in these processes using mutagenesis, whole cell electrophysiology, and molecular dynamics simulations and found that the neutralization of four conserved residues, namely Glu(1077) and Asp(1080)-Asp(1082) in human TRPA1, had strong effects on the Ca(2+)- and voltage-dependent potentiation and/or inactivation of agonist-induced responses. The surprising finding was that truncation of the C terminus by only 20 residues selectively slowed down the Ca(2+)-dependent inactivation 2.9-fold without affecting other functional parameters. Our findings identify the conserved acidic motif in the C terminus that is actively involved in TRPA1 regulation by Ca(2+).

• MeSH
• buněčné linie MeSH
• kationtové kanály TRP chemie   metabolismus   fyziologie   MeSH
• kationtový kanál TRPA1 MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• proteiny nervové tkáně chemie   metabolismus   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• vápníkové kanály chemie   metabolismus   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRP MeSH
• kationtový kanál TRPA1 MeSH
• proteiny nervové tkáně MeSH
• TRPA1 protein, human MeSH Prohlížeč
• vápník MeSH
• vápníkové kanály MeSH

The ankyrin transient receptor potential channel TRPA1 is a non-selective cationic channel that is expressed by sensory neurons, where it can be activated by pungent chemicals, such as AITC (allyl isothiocyanate), cinnamon or allicin, by deep cooling (<18 °C) or highly depolarizing voltages (>+100 mV). From the cytoplasmic side, this channel can be regulated by negatively charged ligands such as phosphoinositides or inorganic polyphosphates, most likely through an interaction with as yet unidentified positively charged domain(s). In the present study, we mutated 27 basic residues along the C-terminal tail of TRPA1, trying to explore their role in AITC- and voltage-dependent gating. In the proximal part of the C-terminus, the function-affecting mutations were at Lys969, Arg975, Lys988 and Lys989. A second significant region was found in the predicted helix, centred around Lys1048 and Lys1052, in which single alanine mutations completely abolished AITC- and voltage-dependent activation. In the distal portion of the C-terminus, the charge neutralizations K1092A and R1099A reduced the AITC sensitivity, and, in the latter mutant, increased the voltage-induced steady-state responses. Taken together, our findings identify basic residues in the C-terminus that are strongly involved in TRPA1 voltage and chemical sensitivity, and some of them may represent possible interaction sites for negatively charged molecules that are generally considered to modulate TRPA1.

• MeSH
• aminokyseliny bazické genetika   fyziologie   MeSH
• ankyrinová repetice MeSH
• ankyriny MeSH
• ionty farmakologie   MeSH
• kationtové kanály TRP chemie   metabolismus   MeSH
• kationtový kanál TRPA1 MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• nervové receptory chemie   MeSH
• proteiny nervové tkáně chemie   metabolismus   MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• vápníkové kanály chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny bazické MeSH
• ankyriny MeSH
• ionty MeSH
• kationtové kanály TRP MeSH
• kationtový kanál TRPA1 MeSH
• proteiny nervové tkáně MeSH
• TRPA1 protein, human MeSH Prohlížeč
• vápníkové kanály MeSH

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.

• MeSH
• elektrofyziologie metody   MeSH
• iontové kanály chemie   MeSH
• kationtové kanály TRP metabolismus   MeSH
• kationtové kanály TRPV metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze cílená MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• terciární struktura proteinů MeSH
• vysoká teplota MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• iontové kanály MeSH
• kationtové kanály TRP MeSH
• kationtové kanály TRPV MeSH
• membránové proteiny MeSH