The interplay of SK3, a Ca²⁺ sensitive K⁺ ion channel, with Orai1, a Ca²⁺ ion channel, has been reported to increase cytosolic Ca²⁺ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K⁺ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca²⁺ levels that decrease SK3 K⁺ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3–Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3–Orai1 interplay by significantly decreasing their co-localization. Forced STIM1–Orai1 activity and associated Ca²⁺ influx promote SK3 K⁺ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.

Orai1, the Ca²⁺-selective pore in the plasma membrane, is one of the key components of the Ca²⁺release-activated Ca²⁺ (CRAC) channel complex. Activated by the Ca²⁺ sensor in the endoplasmic reticulum (ER) membrane, stromal interaction molecule 1 (STIM1), via direct interaction when ER luminal Ca²⁺ levels recede, Orai1 helps to maintain Ca²⁺ homeostasis within a cell. It has already been proven that the C-terminus of Orai1 is indispensable for channel activation. However, there is strong evidence that for CRAC channels to function properly and maintain all typical hallmarks, such as selectivity and reversal potential, additional parts of Orai1 are needed. In this review, we focus on these sites apart from the C-terminus; namely, the second loop and N-terminus of Orai1 and on their multifaceted role in the functioning of CRAC channels.

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.

The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP₂). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP₂ in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP₂ depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.