Journal: Frontiers in Physiology

Article Title: Contributions of Ca V 1.3 Channels to Ca 2+ Current and Ca 2+ -Activated BK Current in the Suprachiasmatic Nucleus

doi: 10.3389/fphys.2021.737291

Figure Lengend Snippet: Effects of Ca 2+ chelators and Ca 2+ channel inhibitors on BK currents recorded from SCN neurons during the day and night. Paxilline-sensitive macroscopic BK currents were recorded from C57BL6 WT (A–E) and Ca V 1.3 WT (F) SCNs. All intracellular solutions in this study were made with 0.9 mM EGTA, except where 5 mM BAPTA was substituted (A–E) . Currents were elicited from a holding potential of −90 mV by 150-ms voltage steps from −110 to +90 mV in +20-mV increments. (A,B) Representative BK currents from −90 to +90 mV are shown from day (A) and night (B) SCN neurons. (C,D) Current-voltage plot comparing BK current density recorded in either control EGTA or BAPTA during the day (C) and night (D) . (E) Summary of BK current density at +90 mV recorded with control EGTA or BAPTA with Ca 2+ channel inhibitors 10 μM nimodipine (Nim) during the day, or 10 μM dantrolene (Dan) at night. In EGTA, Nim decreased BK currents compared to controls ( P = 0.0008), with no significant difference in BAPTA. At night, BK currents were decreased with BAPTA compared to control EGTA conditions ( P < 0.0001). Then Dan decreased BK currents in control EGTA ( P = 0.001), but BAPTA was not significant. (F) Ca 2+ channel inhibitor sensitivity in Ca V 1.3 WT SCN neurons. Summary of BK current density at +90 mV recorded with EGTA in control (no drug) conditions, or in the presence of Nim or Dan in day and night. Ca V 1.3 WT BK currents were decreased in Nim during the day compared to controls ( P = 0.01), but not Dan. At night, Ca V 1.3 WT BK currents were decreased in Dan compared to control ( P = 0.008) and Nim ( P < 0.0001), but Nim and control were not different. * P < 0.05, One-way ANOVA with Bonferroni's post hoc test between all conditions within day or night. N's represent individual cells recorded from C57BL6 WT (EGTA, BAPTA) day: control (11 neurons, three slices; six neurons, two slices); Nim (seven neurons, two slices; four neurons, one slice) and night: control (12 neurons, three slices; seven neurons, two slices); Dan (five neurons, two slices; three neurons, one slice). Ca V 1.3 WT (day, night): control (11 neurons, six slices; 12 neurons, three slices), Nim (14 neurons, four slices; 13 neurons, four slices), and Dan (five neurons, two slices; seven neurons, two slices). Data are mean ± SEM.

Article Snippet: Drugs used in these experiments were: L-type Ca 2+ channel inhibitor nimodipine (Nim, 10 μM, Alomone Labs, Jerusalem, Israel, #N-150), Ryanodine Receptor inhibitor dantroline (Dan, 10 μM, Sigma, #D9175), BK current inhibitor Paxilline (Pax, 10 μM, Alomone Labs, Jerusalem, Israel, #P-450) and Sodium channel inhibitor tetrodotoxin (TTX, 1 μM, Alomone Labs, Jerusalem, Israel, #T-550).

Techniques:

Journal: Frontiers in Physiology

Article Title: Contributions of Ca V 1.3 Channels to Ca 2+ Current and Ca 2+ -Activated BK Current in the Suprachiasmatic Nucleus

doi: 10.3389/fphys.2021.737291

Figure Lengend Snippet: Nimodipine-sensitive Ca 2+ currents from Ca V 1.3 WT and Ca V 1.3 KO SCN during the day and night. Macroscopic Ca 2+ currents were elicited from a holding potential of −90 mV by 150-ms voltage steps from −90 to +50 mV in +10-mV increments. Nimodipine-sensitive LTCC currents were isolated by subtracting currents obtained in 10 μM nimodipine from the total cell current. (A,E) Representative nimodipine-sensitive (Nim-sens) current traces from Ca V 1.3 WT (A) and Ca V 1.3 KO (E) neurons during the day (top current traces) and night (bottom). (B,C) Current-voltage plot for Ca V 1.3 WT Ca 2+ currents before nimodipine (total) and the nimodipine-sensitive (Nim-sens) current from day (B) and night (C) neurons. (D) Comparison of Ca V 1.3 WT nimodipine-sensitive normalized current density between day and night. Ca V 1.3 WT nimodipine-sensitive currents were larger during the day (at −10 mV) compared to night (at 0 mV) ( P = 0.01). (E) Representative nimodipine-sensitive currents from Ca V 1.3 KO neurons during the day (top current traces) and night (bottom). (F,G) Current-voltage plot of Ca V 1.3 KO total and nimodipine-sensitive currents from day (F) and night (G) neurons. (H) Comparison of Ca V 1.3 KO nimodipine-sensitive normalized current density between day and night. Ca V 1.3 KO nimodipine-sensitive currents were not different between day (at 0 mV) and night (at −10 mV) ( P = 0.2). (I,J) Comparisons of nimodipine-sensitive current densities from Ca V 1.3 WT and Ca V 1.3 KO SCN during the day (I) and night (J) . Ca V 1.3 KO nimodipine-sensitive currents were smaller than Ca V 1.3 WT currents during the day ( P = 0.009) but not at night ( P = 0.5). P < 0.05, unpaired Welch's t tests. N's represent individual cells recorded from Ca V 1.3 WT (eight neurons, three slices day; 10 neurons, four slices night) and Ca V 1.3 KO (12 neurons, two slices day; nine neurons, one slice night). Data are mean ± SEM.

Article Snippet: Drugs used in these experiments were: L-type Ca 2+ channel inhibitor nimodipine (Nim, 10 μM, Alomone Labs, Jerusalem, Israel, #N-150), Ryanodine Receptor inhibitor dantroline (Dan, 10 μM, Sigma, #D9175), BK current inhibitor Paxilline (Pax, 10 μM, Alomone Labs, Jerusalem, Israel, #P-450) and Sodium channel inhibitor tetrodotoxin (TTX, 1 μM, Alomone Labs, Jerusalem, Israel, #T-550).

Techniques: Isolation

Journal: Frontiers in Physiology

Article Title: Contributions of Ca V 1.3 Channels to Ca 2+ Current and Ca 2+ -Activated BK Current in the Suprachiasmatic Nucleus

doi: 10.3389/fphys.2021.737291

Figure Lengend Snippet: BK currents from Ca V 1.3 WT and Ca V 1.3 KO SCN during the day and night. Paxilline-sensitive macroscopic BK currents were recorded as in . (A) Summary of BK current densities at +90 mV recorded from Ca V 1.3 WT and Ca V 1.3 KO neurons during the day and night. BK currents were increased at night for both Ca V 1.3 WT ( P = 0.004) and Ca V 1.3 KO ( P = 0.007) compared to the day. * P < 0.05, unpaired Welch's t tests. (B) Percentage of SCN neurons with BK current in Ca V 1.3 WT and Ca V 1.3 KO neurons recorded in control conditions or in 10 μM nimodipine (Nim) during the day and night. The number of neurons exhibiting BK currents was not different between Ca V 1.3 WT and Ca V 1.3 KO ( P = 0.14, Fischer's exact test). N's: Ca V 1.3 WT, 12/13 (cell with BK current/total number recorded); Ca V 1.3 WT Nim, 10/14; Ca V 1.3 KO, 7/11 and night: Ca V 1.3 WT, 12/12; Ca V 1.3 WT Nim, 13/13; Ca V 1.3 KO, 6/6. (C,D) Current-voltage plot for normalized BK current densities from Ca V 1.3 WT and Ca V 1.3 KO neurons in control conditions (C) and after application of Nim (D) during the day. Ca V 1.3 WT BK currents were increased at +90 mV compared to Ca V 1.3 KO in control conditions ( P = 0.03) but not in Nim ( P = 0.6). * P < 0.05, Repeated measures ANOVA with Bonferroni's post hoc test between WT and KO across −10 to +90 mV. (E) Current-voltage plot for BK current densities from Ca V 1.3 WT and Ca V 1.3 KO at night, in control and after application of Nim. Ca V 1.3 KO BK currents were increased at +30 ( P = 0.007), +50 ( P = 0.002) and +70 mV ( P = 0.01) compared to Ca V 1.3 WT in control conditions at night. N's represent individual cells recorded in (day, night): Ca V 1.3 WT (13 neurons, six slices; 12 neurons, three slices), Ca V 1.3 WT Nim (14 neurons, four slices; 13 neurons, four slices), and Ca V 1.3 KO (11 neurons, four slices; six neurons, two slices), and Ca V 1.3 KO Nim (three neurons, one slice; three neurons, two slices). Data are mean ± SEM.

Article Snippet: Drugs used in these experiments were: L-type Ca 2+ channel inhibitor nimodipine (Nim, 10 μM, Alomone Labs, Jerusalem, Israel, #N-150), Ryanodine Receptor inhibitor dantroline (Dan, 10 μM, Sigma, #D9175), BK current inhibitor Paxilline (Pax, 10 μM, Alomone Labs, Jerusalem, Israel, #P-450) and Sodium channel inhibitor tetrodotoxin (TTX, 1 μM, Alomone Labs, Jerusalem, Israel, #T-550).

Techniques: