TRPV6 Regulation by Cis-22a and Cholesterol

Abstract

The highly calcium-selective transient receptor potential vanilloid-type channel TRPV6 is important for epithelial Ca2+ transport. Proper regulation of the inherently constitutively active TRPV6 channels is intricate in preserving Ca2+ homeostasis, whereby structural and functional data suggest that lipids hold an essential role. Altered expression levels or specific TRPV6 mutations may lead to diseases, hence, TRPV6 represents an interesting target for pharmacological modulation. Recent cryo-EM data identified that the specific TRPV6 blocker cis-22a binds, apart from the pore, to a site within the tetrameric channel that largely matches a lipid binding pocket, LBS-2. Therein, cis-22a may replace a lipid such as cholesterol that is bound in the open state. Based on site-directed mutagenesis and functional recordings, we identified and characterized a series of residues within LBS-2 that are essential for TRPV6 inhibition by cis-22a. Additionally, we investigated the modulatory potential of diverse cholesterol depletion efforts on TRPV6 activity. While LBS-2 mutants exhibited altered maximum currents, slow Ca2+-dependent inactivation (SCDI) as well as less inhibition by cis-22a, TRPV6 activity was resistant to cholesterol depletion. Hence, lipids other than cholesterol may predominate TRPV6 regulation when the channel is expressed in HEK293 cells.

Bidirectional Allosteric Coupling between PIP2 Binding and the Pore of the Oncochannel TRPV6

“…Of note, in all our analysis, levels of inhibition were considerably higher when derived from electrophysiological measurements compared to Ca 2+ imaging experiments, what is interestingly a common observation when comparing inhibitory levels yielded from different methods [ 47 , 49 , 62 ]. In this regard, it needs to be emphasized that the patch clamp technique directly monitors Ca 2+ influx via TRPV6 channels, whereas in Ca 2+ imaging experiments, resultant changes in intracellular Ca 2+ levels are yielded from a fluorescent read-out, which are impacted by various cellular processes, as well as dependent on the experimental conditions.…”

“…In view of the role for channel activation, it is somewhat puzzling that both the removal (TRPV6 R470A, TRPV6 K484A, TRPV6 R492Q) and insertion (TRPV6 G488R) of positive charges within LBS-2 apparently reduced sensitivity to PIP 2 depletion and that the TRPV6 G488R substitution, which reportedly increases affinity to the activating lipid PIP 2 [ 59 ], leads to experimental detection of smaller currents. Interestingly, the latter effect has already been observed by Velisetty and colleagues (2016) and is likely related to the lacking inactivation of the TRPV6 G488R mutant [ 59 , 62 ], leading to Ca 2+ overload and cell death if expression levels are too high. For some of the analyzed LBS-2 residues, previous experimental data (K484) [ 58 ] and the aforementioned structural model of PIP 2 -bound TRPV6 (R492) indicate their involvement in PIP 2 binding.…”

“…In the case of TRPV6, cryo-EM data indicate that linker residues contribute to LBS-2; thus, they are apparently involved in lipidic regulation [ 46 ] and part of one of the two binding sites of the small molecule inhibitor cis-22a [ 47 ] ( Figure 2 C). In the latter regard, we have shown in one of our recent publications that the TRPV6 channel becomes largely insensitive to the mentioned inhibitor upon inserting the G488R mutation into the TM4-TM5 linker [ 62 ]. Here, we again observed that TRPV6 G488R currents are not inhibited by the application of 0.1 µM cis-22a ( Figure 2 D), which equals approximately the IC 50 of 82 nM of this blocker [ 47 ].…”

“…TRPV6 LBS-2 mutants which showed complete unresponsiveness to cis-22a treatment in the NFAT screen were consequently characterized more closely in patch clamp measurements. Thereby, some mutants which were investigated in our previous publications [ 47 , 62 ] are shown again to simplify comparison with other treatments. Current inhibition of TRPV6 R470A [ 47 ], TRPV6 K484A [ 62 ], and TRPV6 R492Q was significantly reduced upon applying 0.1 μM cis-22a.…”

“…Thereby, some mutants which were investigated in our previous publications [ 47 , 62 ] are shown again to simplify comparison with other treatments. Current inhibition of TRPV6 R470A [ 47 ], TRPV6 K484A [ 62 ], and TRPV6 R492Q was significantly reduced upon applying 0.1 μM cis-22a. Successive perfusion of 10 μM cis-22a, a dose which almost completely (~98%) impedes TRPV6 WT currents, also culminated in a significantly reduced inhibition of the mutants ( Figure 4 C), which is in line with the data on NFAT localization.…”