Allam S , Levenson-Palmer R , Chia Chang Z , Kaur S , Cernuda B , Raman A , Booth A , Dobbins S , Suppa G , Yang J , Buraei Z .

R01 GM085234 NIGMS NIH HHS , R01 NS053494 NINDS NIH HHS , R15 GM124013 NIGMS NIH HHS

             neurotransmitter secretion
             cell migration

                atrial fibrillation
                Alzheimer's disease
                epilepsy
                schizophrenia
                migraine

	FIGURE 1. A chimeric P/Q-T channel insensitive to Gem has slow inactivation. (A) WT P/Q channels (top) were expressed in Xenopus oocytes with the β3 and α2δ subunits in the presence or absence of Gem and currents were recorded using two-electrode voltage clamp. Gem inhibits P/Q channels (top bar graph). Gem inhibition can be abolished when the IIS1-IIS3 region (dark blue topology in middle panel) is replaced with its counterpart in the Gem insensitive T-channels, which confirms a previous study (Fan et al., 2010). Interestingly, restoring the native 8 amino acid cytosolic PQ-channel region back to this chimera, which gives rise to the PQ(TIIS1-IIS2) chimera in the bottom panel, still yields Gem-insensitive channels (see methods for chimeric amino acids). (B) 1,500 ms long voltage pulses reveal that the PQ(TIIS1-IIS2) channel chimera is slowly inactivating compared to WT channels. Bar graph shows whisker plots of fold inactivation calculated by comparing peak current at 0 mV to leftover current at the end of the 1.5 s pulse. ** indicates p < 0.01.
	FIGURE 2. Different mutations in PQ channels that alter inactivation also alter sensitivity to Gem. (A) Various mutant channels (red dot indicates mutation site) with differing inactivation kinetics (see Supplementary Figure S1) were expressed in Xenopus oocytes and their currents during 0.5 s voltage steps recorded. The vertical bar to the left of the currents indicates 10 µA. Currents were recorded in the presence or absence of Gem but shown here are those in the presence or absence of HA-Gem. In this set of experiments the amount of HA-tagged Gem RNA injected into the oocytes was adjusted to a level that gives ∼50% inhibition in WT P/Q-channels (top bar gaph) so both increases and a decreases in Gem potency can be observed in the mutants. Ten Oocytes were collected after recording for each of the experimental groups in the westerns in (B) which show similar amounts for HA-Gem expression for all conditions. See Methods for HA-Gem quantification.
	FIGURE 3. Coexpression of the Cavβ2a subunit can abolish Gem inhibition of P/Q-channels and Gem delays channel recovery from inactivation. (A) Co-expression of P/Q channels with the β2a subunit, which slows inactivation, abolishes inhibition by Gem (“+Gem” indicates the addition of 0.03 ng/cell of Gem cRNA). Inhibition can be restored using larger amounts of injected Gem cRNA (“+++” bar graphs are for cells with with 0.3 ng of Gem cRNA/cell). (B) Recovery from inactivation was studied using minute long pulses to 0 mV followed by recovery from inactivation at a holding potential of −80 mV. During recovery, short test pulses to 0 mV were given every 10 s (top panel) and currents recorded in the presence or absence of Gem (lower panel). (C) The average time course of recovery from inactivation (note log scale); currents were normalized to post-recovery levels. Smooth lines show an exponential fit to the average time course of recovery (τ = 8.6 s without Gem and 34.3 s with Gem). Bar graphs show standard whisker plots for the time constants, which were significantly different, p < 0.01. (D) A diagram based on Bähring and Covarrubias (2011) helping visualize transitions between closed (C), open (O), and inactivated (I) states. See text. (E) Cells without Gem (upper current panels) or with Gem cRNA (lower current panels), alongside cRNAs for the PQ channel (Cav2.1) pore-forming subunit cRNA, β3, and α2δ subunits were held at −60 mV for 8 min, their currents recorded (left side currents), then switched to a holding potential of −100 mV for 8–10 min, and their currents recorded again (right side currents). The increase in current size was expressed in (F) as a fold increase in current in −100 mV compared to −60 mV.
	FIGURE 4. Human disease mutations dramatically alter sensitivity to Gem inhibition. (A) WT and the indicated disease mutants of human PQ and L-channels were coexpressed with β3 and α2δ subunits. Red dots indicate mutations sites. (B) T. S. mutants show dramatic resistance to Gem inhibition, while PQ channel mutants exhibit increased inhibition. White whisker plots are normalized peak currents from cells without HA-Gem, while black bars reflect fold reduction in peak currents from cells co-expresing HA-Gem. Differences in average current amplitudes were statistically significant at the p < 0.01 level, as indicated by the “**”. Similar results with untagged Gem are in Supplementary Figure S4. (C) Westerns show HA-Gem levels in cell lysates (pooled from 8 oocytes for each condition), and were quantified using ImageJ. N ≥ 8 for each sample.

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