The PKC activator indolactam-V does not induce constitutive Ca²⁺ channel activity in colonic myocytes. (A) PKCα expression in colon. The immunoblot shows increasing loads of total protein (10, 15, and 20 µg in lanes 1–3) from guinea pig colon whole homogenate probed with mouse anti-PKCα monoclonal IgG1. The position of molecular weight markers, which were run alongside, is indicated. The result shown is typical of those obtained from two other independent experiments. (B) Ca_V1.2 expression in guinea pig colon. The immunoblot shows increasing loads of total protein (10, 20, 30, and 40 µg in lanes 1–4) from guinea pig colon whole homogenate probed with rabbit anti-Ca_V1.2 polyclonal antibody. Lane 5 shows cardiac ventricular homogenate (10 µg), which was used as a positive control for Ca_V1.2. The position of molecular weight markers run alongside is indicated. The result is typical of two other experiments. (C) In indolactam-V (a PKC activator; 10 µM; open bar above traces), depolarization (−70 to +10 mV; C, vi) activated a voltage-dependent Ca²⁺ current (I_Ca; v) to evoke a rise in [Ca²⁺] in the subplasma membrane space (ii) and bulk cytoplasm (iii). 1 µM nimodipine (iv) applied from a puffer pipette (C, i, left panel, Nimodipine Pipette) did not alter the subplasma membrane (ii) or bulk average (iii) [Ca²⁺], although it blocked depolarization-evoked I_Ca (v) and the accompanying [Ca²⁺] rise (ii and iii). The break in the record is ∼3 min after which I_Ca and [Ca²⁺] rise partially recovered. A bright field image of the cell is shown (C, i, left panel); see also whole cell electrode (right side). (D) Hyperpolarization (−130 mV; vi) from the resting potential (−70 mV; vi) evoked an increase in [Ca²⁺]_PM (ii) and [Ca²⁺]_c (iii). The PKC activator indolactam-V (10 µM; open bar) was present throughout. 1 µM nimodipine (iv), an inhibitor of voltage-dependent Ca²⁺ channels, was applied by pressure ejection from a puffer pipette (D, i, left panel) but did not reduce the hyperpolarization-evoked [Ca²⁺]_c rise. The small change in [Ca²⁺] (ii and iii) on nimodipine application (iv) occurred because of a slight change in the membrane current (v). Changes in the fluorescence ratio with time (C, ii and iii, and D, ii and iii) are derived from the regions encompassing that part of the cell in TIRF (C, i, and D, i, right-hand panels).