Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells

Abstract

Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Cav). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Cav opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet β-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Cavα1 pore-forming subunits of R-type Cav2.3 and to lesser extent L-type Cavs, while confirming the preferential interactions between Syn-1A with L-type (Cav1.2, Cav1.3) Cavs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Cav2.3, whereas Syn-1A prefers L-type Cavs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Cav channels. Syn-3 KD enhanced Ca2+ currents by 46% attributed mostly to R- and L-type Cavs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Cav activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Cav activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Cavs, but preferring Cav2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS.

Fig 1. Syn-3 co-immunoprecipitates (IP) distinct Ca_vs than Syn-1A in INS-1 cells.

Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca_vα1 subunits (Ca_v1.2, Ca_v1.3, Ca_v2.3 and Ca_v2.2) and auxiliary subunits (α₂δ-1 and β3) and SNAP25 in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in S1 Fig), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca_vs. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca_vs (left lanes). Right lanes show the input lysates. All five experiments probed for the Ca_v α subunits and α₂δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.

Fig 2. Syn-3 preferentially binds Ca_v2.3 while Syn-1A preferentially binds Ca_v1.3.

Representative blots show HEK293 cells co-transfected with Ca_v1.3 (A) or Ca_v2.3 (C) with either Syn-1A or Syn-3, then subjected to co-IP with anti-Syn-1A or Syn-3 antibody. Co-precipitated proteins were identified with the indicated antibodies. Densitometric analysis of the co-precipitated Ca_v1.3 (B) or Ca_v2.3 (D), expressed as percent recovery of total lysate inputs. Values are means±SEM, n = 3; *p<0.05, **p<0.01.

Fig 3. Depletion of Syntaxin 3 in INS-1 cells increased voltage-gated Ca²⁺ currents.

(A) Representative traces showing Ca_v currents recorded in whole-cell mode from control and Syn-3 KD INS-1 cells. (B) Current-voltage relationship of Ca_vs from control (n = 16) and Syn-3 KD (n = 11) INS-1 cells. Currents were normalized to cell capacitance to yield current density. Values are means±SEM. (C) Bar chart shows the maximum increase in current density under stimulation of 10 mV voltage. *p<0.05 for control vs Syn-3 KD (D) Representative Ca_v currents from normal INS-1 cells before and after treatment with nifedipine (10 μM Nif; n = 8), SNX482 (400 nM SNX; n = 9) or ω-Conotoxin GVIA (100 nM Conotoxin; n = 10); their summary analysis (E) of the maximum increase in current densities normalized to the percentage of control (Con). ***p<0.001, **p<0.01 and *p<0.05 compared to control. We then performed another set of experiments (different from A-E) to compare the effects of nifedipine and SNX on Syn-3 KD (H and I) and Control INS-1 cells (F and G). (F) Representative Ca_v currents from control INS-1 cells before (control, n = 25) and after treatment with nifedipine (10 μM Nif) (n = 14) or SNX482 (400 nM SNX) (n = 6); their summary analysis (G) of the maximum increase in current densities normalized to the percentage of control (Con). ***p<0.001; *p<0.05 compared to Control. (H) Representative Ca_v currents of Syn-3 KD INS-1 cells before (Syn-3 KD, n = 11) and after treatment with nifedipine (n = 12) or SNX482 (n = 6); and their summary analysis (I) of the maximum increase in current densities normalized as percentages of the Syn-3 KD cells. *p<0.05 compared to Syn-3 KD. Here, Syn-3 KD Ca_v currents were 148% of Control cells, similar to A and B.

Fig 4. Cytoplasmic Syn-3 domain but not cytoplasmic Syn-1A domain regulates Ca_v currents.

(A) GST-Syn-3 cytoplasmic domain (a.a. 1–263) or GST-Syn-1A cytoplasmic domain (a.a. 1–265) or GST (control) was dialyzed into INS-1 cells, then Ca_v currents recorded. Shown are representative traces in the whole-cell mode with stimulation from −80–60 mV. (B) Current-voltage relationship of Ca_v channels. Currents were normalized to cell capacitance to yield current density. (C) Bar chart showing the maximum current density in INS-1 cells dialyzed with GST control (n = 9), GST-Syn-3 (n = 9), or GST-Syn-1A (n = 8). Values are means±SEM; *p<0.05; NS: no significant difference. (D and E) Representative blots show HEK293 cells transfected with Ca_v1.3 (D) or Ca_v2.3 (E) subjected to pull down with 300 pmol of GST-Syn-1A (a.a. 1–265), GST-Syn-3 (a.a. 1–263) or GST. (F) Summary analysis of four separate experiments. Data was expressed as means ± SEM; *p<0.05.