Figure 3.

C. elegans CLHM-1 is regulated by voltage and extracellular divalent cations. A, Currents observed in oocytes expressing C. elegans CLHM-1 in response to voltage pulses from −120 to +60 mV. The holding potential was at the resting potential (ranged from −5 to −15 mV) in divalent-free bath solution containing 0.5 mm EGTA and 0.5 mm EDTA. B, C, Using the same protocol as in Figure 1B, currents observed in oocytes expressing C. elegans CLHM-1 in standard bath solution containing 5 mm Ca²⁺ (B) or 5 mm Mg²⁺ (C). All oocytes were injected with the Cx38 oligonucleotide and BAPTA to inhibit endogenous currents (Ma et al., 2012); currents were not observed in H₂O-injected oocytes in the presence or absence of divalent cations (Fig. 1C; data not shown). D, Instantaneous current–voltage relationship in divalent-free (black) and 2 mm Ca²⁺ (gray) bath solutions. Tail currents observed after stepping to the different voltages were normalized to average current at the end of the activating +60 mV prepulse; solid lines show linear fit. E, CLHM-1 currents at −80 mV were measured after a series of voltage steps (as in A, B) to determine conductance–voltage relationships in the absence and presence of Ca_o²⁺. The currents in various Ca_o²⁺ solutions were normalized to G_max determined by fitting 0 mm Ca_o²⁺ data with a Boltzmann function for each oocyte. All normalized data were then fit with Boltzmann functions (lines) as for human CALHM1 (Ma et al., 2012) with the assumption that Ca_o²⁺ does not affect G_max. This fit gave rise to the apparent conductance–voltage relationship. Half-activation voltages are as follows: 0 mm Ca_o²⁺ (black squares), V_0.5 = −6.8 ± 0.4 mV; 0.1 mm Ca_o²⁺ (light gray circles), V_0.5 = 24.1 ± 0.4 mV; 0.5 mm Ca_o²⁺ (dark gray diamonds), V_0.5 = 41.4 ± 0.3 mV; 2 mm Ca_o²⁺ (black circles), V_0.5 = 66 ± 0.4 mV; 5 mm Ca_o²⁺ (light gray squares), V_0.5 = 69.1 ± 0.5 mV. n ≥ 5 oocytes for each condition. Error bars show the SE of normalized data.