![Figure 1. Effects of purified human brain CaM on the rat olfactory CNG channel (formed by subunit A2) and the mouse TMEM16A channel. All experiments were excised inside-out patch recordings. (A) Equivalent circuit showing that when membrane resistance (Rm) is reduced, the membrane voltage Vm is shunted away via a current flow through Rm (red arrow). Thus, the measured voltage (εm) is smaller than Vm in absolute values. (B) Effects of the purified human brain CaM on the olfactory CNG channel. Solutions were as those in Fig. 3 of Yu et al. (2014b), except that the total [Ca2+] in the 2-µM cGMP solution is 0.15 mM. Time constants of the CaM inhibition: 46 s (0.5 µM) and 6 s (5 µM). (C–E) Effects of the purified human brain CaM on TMEM16A’s anion permeability. Solutions contained 0.1 mM EGTA and total [Ca2+] of 0.12 mM (C), 0.105 mM (D), or 0.25 mM (E) throughout the recordings. The recordings started in symmetrical 140 mM NaCl (EI=0 = 0 mV). The intracellular solution was then changed to a solution containing 130 mM NaHCO3 plus 10 mM NaCl, during which 5 µM CaM was applied (red horizontal line). The values of EI=0 in CaM minus that without CaM (ΔV) were 0.2–5.9 mV (n = 6), 8.3–20.5 mV (n = 4), and −0.4 to 1.8 mV (n = 5) in C, D, and E, respectively. Notice that as total [Ca2+] is increased, the CaM effect is reduced. (F) Effects of the purified human brain CaM shown in C and D were likely caused by a reduction of the intracellular free [Ca2+]. Experimental solutions were the same as those in C except that 90 µM EDTA (instead of CaM) was added. ΔV = 16, 18, and 38 mV from three patches. Bottom panels: Voltage-clamp experiments (black, 40 mV; red, −40 mV) with the bath solutions being changed from the 0-Ca2+ solution (140 mM NaCl) to the HCO3− solutions (horizontal blue line) used in the current-clamp experiment as indicated by arrows. Notice that the solution containing an extra 90 µM EDTA did not activate detectable current. (G) The purified human brain CaM did not alter EI=0 in the solution used in Jung et al. (2013)—namely, 130 mM NaHCO3, 20 mM NaCl, 10 mM EGTA, and ∼9.8 mM total [Ca2+] (calculated free [Ca2+] = ∼3 µM).](https://cdn.rupress.org/rup/content_public/journal/jgp/145/1/10.1085_jgp.201411294/5/jgp_201411294r_fig1.jpeg?Expires=1767772311&Signature=tHoYv2y2AgHiFpPw4j4jJLhL1BvvjltQPC9Me0jhjZIeGAA0IJ8GJN14BjJeST5vSlfBT5BoWPMcngsAl5sVRhTdP1vrZCCC0liR-k2EjWvXTkecFyVJ9a4s8Vf01XjQeXjF~sH-uxmgOAlqgTEuKVSIm-ZAX4tgZK2f5tkA3-n5py1yTXES5QcEnFJk88jsfGL~POI7leH1yL-baSZDnk4LrCtYRFvXC0i~M6XnCfFXVtHaejVVluCf1AQ6HLAHSTrqPz2Y5WuwBWrm7Js1LBZZp-HjOkk1rFP2EjWMtxQW5gPH1Ro4RtvFPpg~d8jBlq5Hf-3j7D~U1K~cfrra8A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effects of purified human brain CaM on the rat olfactory CNG channel (formed by subunit A2) and the mouse TMEM16A channel. All experiments were excised inside-out patch recordings. (A) Equivalent circuit showing that when membrane resistance (Rm) is reduced, the membrane voltage Vm is shunted away via a current flow through Rm (red arrow). Thus, the measured voltage (εm) is smaller than Vm in absolute values. (B) Effects of the purified human brain CaM on the olfactory CNG channel. Solutions were as those in Fig. 3 of Yu et al. (2014b), except that the total [Ca2+] in the 2-µM cGMP solution is 0.15 mM. Time constants of the CaM inhibition: 46 s (0.5 µM) and 6 s (5 µM). (C–E) Effects of the purified human brain CaM on TMEM16A’s anion permeability. Solutions contained 0.1 mM EGTA and total [Ca2+] of 0.12 mM (C), 0.105 mM (D), or 0.25 mM (E) throughout the recordings. The recordings started in symmetrical 140 mM NaCl (EI=0 = 0 mV). The intracellular solution was then changed to a solution containing 130 mM NaHCO3 plus 10 mM NaCl, during which 5 µM CaM was applied (red horizontal line). The values of EI=0 in CaM minus that without CaM (ΔV) were 0.2–5.9 mV (n = 6), 8.3–20.5 mV (n = 4), and −0.4 to 1.8 mV (n = 5) in C, D, and E, respectively. Notice that as total [Ca2+] is increased, the CaM effect is reduced. (F) Effects of the purified human brain CaM shown in C and D were likely caused by a reduction of the intracellular free [Ca2+]. Experimental solutions were the same as those in C except that 90 µM EDTA (instead of CaM) was added. ΔV = 16, 18, and 38 mV from three patches. Bottom panels: Voltage-clamp experiments (black, 40 mV; red, −40 mV) with the bath solutions being changed from the 0-Ca2+ solution (140 mM NaCl) to the HCO3− solutions (horizontal blue line) used in the current-clamp experiment as indicated by arrows. Notice that the solution containing an extra 90 µM EDTA did not activate detectable current. (G) The purified human brain CaM did not alter EI=0 in the solution used in Jung et al. (2013)—namely, 130 mM NaHCO3, 20 mM NaCl, 10 mM EGTA, and ∼9.8 mM total [Ca2+] (calculated free [Ca2+] = ∼3 µM).
