Relevant Pharmacology of Sodium, Calcium, and Channels

Otilonium Bromide Inhibits Calcium Entry Through L-Type Calcium Channels

Figure 1.1.

Block of native inward L-type Ca2+ current by otilonium bromide (OB). Panel A shows representative Ca2+ current trace families recorded in NMDG-Cl or NaCl Ringer’s solution with 0, 0.09, 0.9 or 9 uM OB. Human jejunal circular smooth muscle cells were patch clamped using the pulse protocol shown in the inset. Panel B displays normalized current–voltage relationships (circle NaCl Ringer, square 0.09, triangle 0.9, and diamond 9 uM OB). Panel C summarizes the mean maximal peak Ca2+ current in cells exposed to 0–9 uM OB (n = 9, *P < 0.05).

Figure 1.2.

Otilonium bromide (OB) and Na+ currents. Representative whole cell Na+ current traces were recorded in NaCl Ringer's solution with 0-9 uM OB (panel A) from a human intestinal circular smooth muscle cell. The pulse protocol is shown in the inset. Panel B displays the normalized current-voltage relationship for peak Na+ current (circle NaCl Ringer, square 0.09, triangle 0.9 and diamond 9 uM OB, n = 7). Panel C summarizes the mean maximal peak Na+ current in cells exposed to 0-9 uM OB. At 9 uM OB there was a trend for Na+ current block but this did not reach statistical significance (n = 7, P > 0.05).

Figure 1.3.

Lack of effect of otilonium bromide (OB) on outward K+ currents. Panel A shows representative whole cell K+ current recordings in NaCl Ringer’s solution with 0-9 uM OB from a human intestinal circular smooth muscle cell. Current at +10 mV (predominantly delayed rectifier K+ channels, DR) or +120 mV (predominantly Ca2+-activated K+ channels, KCa) are shown in black, whereas all other traces are shown in grey. The pulse protocol is shown in the inset. Panel B displays the normalized current-voltage relationship for K+ current averaged from 200 to 400 ms after the start of the pulse (n = 5). Panel C summarizes the average K+ current at +10 and +120 mV in cells exposed to 0-9 uM OB (n = 6, P > 0.05).

Figure 1.4.

Otilonium bromide (OB) inhibits heterologously expressed L-type Ca2+ channel subunits. The pore-forming alpha-1C and auxiliary beta-2 L-type Ca2+ channel subunits were expressed in HEK293 cells, and representative whole cell Ca2+ currents were recorded in normal Ringer’s solution with 0-9 uM OB (panel A). The pulse protocol is shown in the inset. Panel B shows the normalized current-voltage relationship for peak Ca2+ current (circle NaCl Ringer, square 0.09, triangle 0.9 and diamond 9 uM OB, n = 6). Panel C summarizes the mean maximal peak Ca2+ current in cells exposed to 0-9 uM OB (n = 6, *P < 0.05).

Figure 1.5.

Deletion of the C- and N-termini of the alpha-1C subunit does not block the effect of otilonium bromide on the Ca2+ channel. L-type Ca2+ currents were recorded from HEK293 cells expressing either alpha-1C minus the first 109 N-terminal amino acids plus beta-2 (panel A) or alpha-1C minus C-terminal amino acids distal to 1703 plus beta-2 (panel B). Cells were exposed to 0 or 1 uM OB (A1 and B1) dissolved in normal Ringer solution. The pulse protocol is included in the inset. Normalized current-voltage relationships for peak Ca2+ current for the N-terminal deletion are shown in A2 and for the C-terminal deletion in B2. The mean maximal peak Ca2+ currents with and without 1 uM OB for the N-terminal deletion are shown in panel A3 and for the C-terminal deletion in panel B3. Both illustrate Ca2+ current block by 1 uM OB (*P < 0.05).

Effect of Mibefradil on Sodium and Calcium Currents

Figure 2.1.

Block of expressed T-type Ca2+ channel subunits alphaI3.3b + beta2 by mibefradil. A: representative whole cell T-type Ca2+ channel currents recorded in NaCl Ringer solution with 0, 0.1, 1, or 10 uM mibefradil. B: current-voltage relationship for peak inward T-type Ca2+ current was normalized to the maximum peak inward current of the control record. C: summary of the mean normalized peak T-type Ca2+ current in cells exposed to 0-10 uM mibefradil (n = 6 per concentration, *P < 0.05).

Figure 2.2.

Block of expressed L-type Ca2+ channel subunits alpha-1C + beta-2 by mibefradil. A: representative whole cell L-type Ca2+ channel currents recorded in NaCl Ringer’s solution with 0, 1, or 10 uM mibefradil. B: current-voltage relationship for peak inward L-type Ca2+ current was normalized to the maximum peak inward current of the control record. C: summary of the mean normalized peak L-type Ca2+ current in cells exposed to 0-10 uM mibefradil (n = 5 per concentration, *P < 0.05).

Figure 2.3.

Block of Na+ channel alpha-subunit SCN5A by mibefradil. A: representative whole cell Na+ channel currents recorded in NaCl Ringer’s solution with 0, 1, or 10 uM mibefradil. B: current-voltage relationship for peak inward Na+ current was normalized to the maximum peak inward current of the control record. C: summary of the mean normalized peak Na+ current in cells exposed to 0-10 uM mibefradil (n = 6 per concentration, *P < 0.05).

Figure 2.4.

Dose-response curves representing mibefradil inhibition of Na+ and Land T-type Ca2+ channels. Summary of mibefradil concentration vs. normalized peak currents as shown in Figs. 1C, 2C, and 3C. IC50 values for T-type Ca2+, Na+, and L-type Ca2+ channels were (in uM) 0.29 (alpha-I3.3b + beta-2, n = 6), 0.98 (SCN5A, n = 6), and 2.7 (alpha-1C + beta-2, n = 5), respectively.