Reply to Yao et al.: Is the pore turret just thermoTRP channels’ appendix?

Abstract

Numerous studies of thermosensitive transient receptor potential (thermoTRP) channels have focused on the outer pore that contains sites critical for the channel's sensitivity to temperature, acidification, spider toxin, and other gating modulators (Fig. 1). Two segments of each subunit contribute to the outer pore structure: one is between S5 and the pore helix, the other is between the selectivity filter and S6. For the convenience of discussion, we call them the large turret and the small turret, respectively. From the available structural information of tetrameric channels, it is expected that the two turrets interact extensively. Indeed, the sites affecting gating spread across the outer pore landscape. Of particular interests are a number of sites that when mutated, lead to permanent opening or closing of the temperature-activation gate (1-3).In agreement with these findings, our study demonstrated that the large turret is part of the temperature-activation pathway (4). This conclusion is based on experimental results that provided thermodynamic, functional, and structural information. Yao and colleagues (5) question our interpretation of one of these experiments, in which changing the large turret structure by sequence replacement yielded functional channels with no apparent temperature gating. Because the mutant channels exhibited near-normal permeation properties and capsaicin activation, structural perturbation is not nonspecific but limited to the temperature gating process-that is to say, the large turret is important for temperature gating. This interpretation is consistent with the observation that site-directed fluorescence recordings revealed large turret movement during heat activation but not capsaicin activation (4).Yao and colleagues object to this interpretation because they recorded temperature-induced whole-cell currents from a rat TRPV1 deletion mutant of a very similar region of the large turret (5). What remains unknown is the relative amplitude of these currents to the capsaicin-induced current from the same cell. In our mouse TRPV1 replacement mutant, there was a tiny heat response [figure 3b of our original report (4)] that, compared to the capsaicin-induced current, represents a very low open probability. The most straightforward interpretation of this phenomenon is that temperature activation is impaired. Interestingly, reduction in heat response was previously reported by the same group in mutant channels generated from the deletion background (6). They noticed "a large irreversible reduction of the capsaicin current after heating." The selective impairment of temperature sensitivity observed in both our study and that of Yao et al. (6) may be mechanistically related.One important but often overlooked issue is that the activity of all ion channels (as well as nonchannel proteins) exhibit temperature sensitivity. The universal temperature-dependence of channel gating, having a Q 10 value ranging from 3 to 7 in most cases, reflects a gain in kinetic energy upon heating that favors p...

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

“…The study attributed the functional effect of the point mutation to be mainly on the channel’s pH sensitivity. If that is the case, the behavior of T633A would suggest that heat activation of the turret deletion mutant might be already changed ( Yang et al, 2010a ). Indeed, given that point mutation and chemical modification of single cysteines in the corresponding region of the mouse TRPV1 channel ( Yang et al, 2010b ) and the neighboring region of the rat TRPV1 channel ( Grandl et al, 2010 ) exhibited substantial gating effects on heat activation, it seems most likely the more dramatic deletion mutation would also affect heat activation.…”

Mutagenesis and Temperature-Sensitive Little Machines

“…However, the striking results presented by Yang et al have been severely questioned by the group of Qin who showed that deletions of the entire turret region are not affecting temperature sensitivity. Clearly this controversy has to be solved [26,27].…”