Figure 5. CALHM3 is an Essential Component of the Voltage-gated Non-selective ATP-release Channel in Type II TBCs.

(A) Representative families of whole-cell currents from WT, Calhm1^–/– and Calhm3^–/– type II TBCs, evoked by 500-ms voltage pulses from -80 mV to +150 mV in 10-mV increments from a holding potential of -40 mV. Dashed line: zero-current level. Non-selective voltage-gated currents are abolished in cells lacking either CALHM1 or CALHM3. Residual currents previously observed in Calhm1^–/– type II TBC (Taruno et al., 2013) were non-specific leak currents likely caused by longer voltage pulses (1 s) used.

(B) G-V relations obtained by whole-cell capacitance-normalized currents at -80 mV evoked by 500-ms voltage pre-pulses. Solid line represents a Boltzmann function fit to WT data (V_0.5 +78.0 ± 8.3 mV, Z₀ 0.88 ± 0.04 e, n = 21). Cell capacitance: 5.0 ± 0.4 pF (n = 21), 4.7 ± 0.7 pF (n = 10) and 4.9 ± 0.3 (n =20) for WT, Calhm1^–/– and Calhm3^–/– cells, respectively. Whole-cell capacitances are not different (two-tailed Student's unpaired t-test: Calhm1^–/– versus WT, P = 0.633, t₂₉ = 0.841; Calhm3^–/– versus WT, P = 0.226, t₃₉ = 0.226.

(C) Representative families of normalized outward currents from a WT type II TBC and a N2a cell co-expressing CALHM1+CALHM3, evoked by 500-ms voltage pulses from -80 mV to +120 mV in 10-mV increments from a holding potential of -40 mV. Dashed line: zero-current level.

(D) Activation-time constants τ_fast (lower panel) and τ_slow (upper panel) obtained from double-exponential fits to activation currents of WT type II TBC (n = 21) and N2a cells co-expressing CALHM1+CALHM3 (n = 22), respectively. Heterologous expression of CALHM1 with CALHM3 in N2a cells generated currents with voltage-dependent kinetic features identical to those of type II TBCs. Mean ± s.e.m.

(E) Carbenoxolone (CBX) inhibits non-selective voltage-gated currents in type II TBC (n = 9) (top) and CALHM1+3 currents in N2a cells (n = 14) (bottom). Representative families of whole-cell currents in a WT type II TBC before, after perfusion of 10 μM CBX in bath solution for ∼10 min, and after 5-min wash-out by normal bath solution, evoked by 500-ms voltage pulses from -80 mV to +100 mV in 10-mV increments from a holding potential of -40 mV. Dashed line: zero-current level. Representative families of whole-cell currents in a N2a cell co-expressing CALHM1+CALHM3 before, after perfusion with10 μM CBX for ∼10 min, and 5 min after wash-out by the normal bath solution, evoked by 500-ms voltage pulses from -80 mV to +130 mV in 10-mV increments from a holding potential of -40 mV. Dashed line: zero-current level.

See also Figures S6 and S7.