225-LB: Targeting ß-Cell Endoplasmic Reticulum (ER) TALK-1 Channels to Restore Ca2+ Homeostasis and Reduce ER Stress in Diabetes

Perturbations in Ca²⁺ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K⁺ countercurrent for [Ca²⁺]_ER release, thereby tuning [Ca²⁺]_ER storage and release. During β-cell Ca²⁺ influx, TALK-1-enhanced [Ca²⁺]_ER release leads to activation of the hyperpolarizing Ca²⁺ activated K⁺ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca²⁺ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca²⁺ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI⁺) flux assay (z’ =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca²⁺]_ER release (e.g. K2P16-i7(IC₅₀=5.3μM); 63.36% [Ca²⁺]_ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca²⁺]_ER storage as well as cytosolic Ca²⁺ oscillations. TALK-1 inhibitors significantly increased islet [Ca²⁺]_ER storage and Ca²⁺ oscillation frequency (e.g.K2P16-i7; [Ca²⁺]_ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca²⁺]_ER AUC=49.7a.u., peaks/min= 0.17, p-value<0.05). Importantly, the inhibitors did not alter [Ca²⁺]_ER and glucose-stimulated Ca²⁺ oscillation frequency in TALK-1 KO islets. In summary, we have identified the first ER TALK-1 selective inhibitors that can not only be utilized to assess the role of TALK-1 in human β-cell Ca²⁺ handling and GSIS, but also potentially reduce ER dysfunction during diabetes pathogenesis.