Key points Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca2+) handling. TSPAN‐7 is enriched in pancreatic islet cells; however, the function of islet TSPAN‐7 has not been identified. Here, we characterize how β‐cell TSPAN‐7 regulates Ca2+ handling and hormone secretion. We find that TSPAN‐7 reduces β‐cell glucose‐stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose‐stimulated insulin secretion. TSPAN‐7 controls β‐cell function through a direct interaction with L‐type voltage‐dependent Ca2+ channels (CaV1.2 and CaV1.3), which reduces channel Ca2+ conductance. TSPAN‐7 slows activation of CaV1.2 and accelerates recovery from voltage‐dependent inactivation; TSPAN‐7 also slows CaV1.3 inactivation kinetics. These findings strongly implicate TSPAN‐7 as a key regulator in determining the set‐point of glucose‐stimulated Ca2+ influx and insulin secretion. Abstract Glucose‐stimulated insulin secretion (GSIS) is regulated by calcium (Ca2+) entry into pancreatic β‐cells through voltage‐dependent Ca2+ (CaV) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling, and thus they may also modulate GSIS. TSPAN‐7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN‐7 is highly expressed in β‐ and α‐cells; however, the function of islet TSPAN‐7 has not been determined. Here, we show that TSPAN‐7 knockdown (KD) increases glucose‐stimulated Ca2+ influx into mouse and human β‐cells. Additionally, mouse β‐cell Ca2+ oscillation frequency was accelerated by TSPAN‐7 KD. Because TSPAN‐7 KD also enhanced Ca2+ entry when membrane potential was clamped with depolarization, the effect of TSPAN‐7 on CaV channel activity was examined. TSPAN‐7 KD enhanced L‐type CaV currents in mouse and human β‐cells. Conversely, heterologous expression of TSPAN‐7 with CaV1.2 and CaV1.3 L‐type CaV channels decreased CaV currents and reduced Ca2+ influx through both channels. This was presumably the result of a direct interaction of TSPAN‐7 and L‐type CaV channels because TSPAN‐7 coimmunoprecipitated with both CaV1.2 and CaV1.3 from primary human β‐cells and from a heterologous expression system. Finally, TSPAN‐7 KD in human β‐cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN‐7 modulation of β‐cell L‐type CaV channels is a key determinant of β‐cell glucose‐stimulated Ca2+ entry and thus the set‐point of GSIS.

Tetraspanin7-mediated inhibition of β-cell voltage-dependent Ca2+ channels regulates glucose-stimulated insulin secretion. Glucose-stimulated insulin secretion (GSIS) is regulated by Ca2+ influx into pancreatic β-cells through voltage-dependent Ca2+ channels (VDCCs). As some tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling they may also modulate GSIS. TSPAN7 is the most abundant TSPAN in mouse and human islets. TSPAN7 autoantibodies are also found in type 1 diabetic serum, suggesting a link between TSPAN7 and diabetes. The aim herein was to determine if TSPAN7 controls β-cell Ca2+ handling and insulin secretion. Immunofluorescence imaging of mouse and human pancreatic slices indicated that TSPAN7 is highly expressed in β- and α-cells. shRNA-mediated TSPAN7 knockdown (KD) in mouse and human β-cells augmented glucose-stimulated Ca2+ influx (by 32.3±9.5% and 33.8±15.1% respectively) as well as KCl-stimulated Ca2+ influx (45 mM; 34.5±9.6%, human). Mouse β-cell Ca2+ oscillation frequency was also accelerated by TSPAN7 KD (by 29.4±10.1%). As TSPANs interact with and regulate VDCCs, we examined the effect of TSPAN7 on VDCC currents. TSPAN7 KD increased peak L-type VDCC currents in mouse and human β-cells (by 4.1±1.3 pA/pF and 2.0±0.9 pA/pF respectively). Heterologous expression of TSPAN7 with L-type VDCCs in HEK293 cells also decreased VDCC currents (CaV1.2: 4.3±1.8 pA/pF; CaV1.3: 17.5±5.4 pA/pF) and reduced KCl-stimulated (50 mM) Ca2+ influx (CaV1.2: 51.0±7.1%; CaV1.3: 28.4±7.5%). Furthermore, TSPAN7 coimmunoprecipitated with CaV1.2 and CaV1.3 from primary human β-cells and when heterologously expressed in HEK293 cells. Finally, TSPAN7 KD in human β-cells increased basal (5.6 mM glucose; 8.3±5.5 ng*hr-1) and stimulated (45 mM KCl+14 mM glucose; 20.4±17.1 ng*hr-1) insulin secretion. These findings reveal that TSPAN7 modulation of β-cell L-type VDCCs limits glucose-stimulated Ca2+ influx and insulin secretion. Disclosure M. Dickerson: None. P. Dadi: None. R.B. Butterworth: None. D. Jacobson: None. Funding American Diabetes Association (1-17-IBS-024 to D.J.); National Institutes of Health (T32DK101003, DK097392, DK115620); Vanderbilt University Medical Center Diabetes Research and Training Center (P60DK20593)