(A) CaV channel complex with α, β, and α2δ subunits. mRNA expression (transcripts per million, TPM) of voltage-gated calcium (CaV) channel β subunits in nematocyte-enriched cells (blue) and non-enriched cells (grey). n = 6, p<0.0001 for cacnb2.1 in nematocytes versus other cells, two-way ANOVA with post-hoc Bonferroni test. (B) Heterologously-expressed nCav channels (Nematostella cacna1a, cacnb2, cacna2d1) inactivated at very negative voltages (estimated Vi1/2 = -101.5 ± 1.6mV, n = 5) versus mammalian orthologues (mCaV, Vi1/2 = -20.9 ± 3.4mV, n = 10). Apparent activation thresholds were the same: nCaV Va1/2 = -9.8 ± 0.3mV, n = 5, mCaV Va1/2 = -10.4 ± 0.5mV, n = 9. Inactivation was measured in response to 1 s pre-pulses from −110 mV to 10 mV with an inter-sweep holding potential of −90 mV. (C) nCaV exhibited slow inactivation with −70 mV holding potential (0.2 Hz stimulation, 5 s inter-pulse interval) that was best fit by two exponential functions with time constants of 10.0 and 369.5 s. n = 6, multiple row two-tailed student’s t-test with significance of p<0.05 by 15 s and p<0.0001 by 500 s. (D) nCav inactivated at −40 mV and quickly recovered at negative holding potentials. n = 7 for nCaV, n = 6 for mCaV. (E) Voltage-gated currents recorded from nCaV or mCaV following a −110 mV pre-pulse, −50 mV pre-pulse (colored), and 20 mV pre-pulse. CaV β subunits were substituted as indicated (mammalian β in red and Nematostella β in blue). Scale bars = 100 pA, 25 ms. (F) Mammalian β shifts nCaV voltage-dependent inactivation to positive voltages. nCaV Vi1/2 = -73.2 ± 1.2mV, n = 6. nCaV + mβ=−16.9 ± 1.9 mV, n = 6. (G) Half maximal inactivation voltage (Vi1/2) for CaV chimeras. p<0.0001 for nCaV versus nCaV + mβ, mCaV versus mCaV + nβ, one-way ANOVA with post-hoc Tukey test. Inactivation was measured in response to pre-pulses from −100 mV to 10 mV with an inter-sweep holding potential of −110 mV to reduce slow inactivation. Data represented as mean ±sem.