Wu F , Quinonez M , DiFranco M , Cannon SC .

R01 AR063128 NIAMS NIH HHS , R01 AR063182 NIAMS NIH HHS

	Figure 1. Coexpression of Stac3 dramatically enhances hCaV1.1 currents in X. laevis oocytes. Each panel shows currents recorded in a cut-open oocyte clamp with 10 mM Ba2+ as the divalent charge carrier. Step depolarizations to −50 mV through 40 mV were applied from a holding potential of −100 mV. Linear leak correction was performed by scaling and subtracting the response to a 20-mV depolarization. (A) Currents from an oocyte injected with RNA for Stac3, α2-δ1b, and β1a subunits; inset shows the same traces at 100×. (B) Currents recorded when α1S (hCaV1.1), α2-δ1b, and β1a subunits were expressed, but no Stac3. At high gain (100×), slow Ca2+ channel currents were detectable in the absence of Stac3. (C) Maximal inward current observed from oocytes injected with Ca2+ channel subunits with Stac3 (closed triangles) or without Stac3 (open circles). Each symbol represents a separate oocyte, and all were recorded from the same batch. (D–F) Large robust currents were consistently observed in oocytes coexpressing Stac3 with hCaV1.1 (WT or HypoPP mutants), α2-δ1, and β1ab subunits.
	Figure 2. Activation of hCaV1.1 currents is hyperpolarized for HypoPP channels. (A and B) The majority of the current recorded from oocytes expressing Stac3, hCaV1.1 α2-δ1b, and β1a subunits was conducted by hCaV1.1, as shown by a block with 10 µM nifedipine (A) or with 4 mM Co2+ (B). Traces show leak-subtracted currents recorded in 10 mM Ba2+ for depolarization to 20 mV from a holding potential of −100 mV. (C) Steady-state I-V relation for Co2+-sensitive current with 10 mM Ba2+ as the charge carrier. Currents were elicited by depolarization from a holding potential of −100 mV. Symbols are mean values, and smooth lines show the Boltzmann fit using the mean values of the parameters. Error bars represent ±SEM. (D) The hyperpolarized shift of activation for HypoPP mutant channels is shown more clearly by plotting relative conductance, G/Gmax = I/[Gmax ∙ (V−Erev)], as a function of test potential.
	Figure 3. Gating charge displacement had a left-shifted voltage dependence for HypoPP mutant channels. (A) Gating charge displacement currents were recorded in 2 mM Co2+ to block ionic currents. Traces show the superposition of leak-subtracted responses for step depolarizations of −100 to 20 mV in 10-mV increments from a holding potential of −100 mV. (B) The “on” gating charge, calculated from the area under the charge displacement currents in A, was normalized to the maximum and plotted as a function of test potential. Smooth curves show Boltzmann functions using mean values for the parameters of the fit to each oocyte, and error bars represent ±SEM. The midpoint of activation for Q/Qmax was left shifted by 19 mV for R528H and by 7.4 mV for R528G compared with WT.
	Figure 4. Larger inward currents, consistent with an anomalous gating pore conductance, were observed for R528H and R528G HypoPP mutant channels. (A) Exemplary traces shown for currents recorded in response to test potentials of −120 to 20 mV in 10-mV increments from a holding potential of −100 mV. At the more depolarized test potentials, the onset of Ba2+ current conducted by the conventional pore of CaV1.1 channels was detected (green traces). No leak subtraction was performed. (B) Isochronal current, measured in the interval of 3–4 ms after the onset of the test pulse (shaded bars in A), is plotted as a function of test potential. Inward currents at hyperpolarized potentials were larger for R528H and R528G than WT. Error bars represent ±SEM. (C) The slope conductance, based on a linear fit of the I-V relation on the interval of −120 to −20 mV, was larger for oocytes expressing R528H and R528G than for WT. Symbols show responses for individual oocytes, and horizontal lines show the mean values.
	Figure 5. The D296K hCaV1.1 subunit conducts Ba2+ poorly and is nonconducting in Ca2+. (A) Steady-state current amplitude recorded in 10 mM external Ba2+ (no added Ca2+) for a series of 250-ms test depolarizations from a holding potential of −100 mV. Current amplitude is greatly reduced for D296K compared with WT channels, but the voltage dependence of activation is similar. (B) In 10 mM external Ca2+ (no Ba2+), no inward current was detected for D296K channels. Subtraction of a linearly scaled leak response to a depolarization from −100 to −80 mV was used to remove the nonspecific leakage current. Error bars represent ±SEM.
	Figure 6. Block of DK/R528H gating pore current by Co2+. (A) Currents recorded from an oocyte expressing DK/R528H channels in Ca2+ (left) or Ca2+ plus Co2+ (right). The oocyte was held at −100 mV, and currents elicited by test pulses from −120 to 20 mV are shown. Capacitance was partially cancelled by the amplifier, but no analogue or digital leak subtraction was performed. (B) Representative steady-state I-V relation for individual oocytes expressing DK/WT, DK/R528H, or DK/R528G. Closed symbols show control response in 6 mM Ca2+; open symbols show control response after bath exchange to 4 mM Ca2+ and 2 mM Co2+. (C) The Co2+-sensitive current was calculated from the difference of responses in 6 mM Ca2+ minus those in 4 mM Ca2+ plus 2 mM Co2+. Symbols show mean with SEM error bars for n = 9 DK/WT-, 9 DK/R528H-, and 6 DK/R528G-expressing oocytes.
	Figure 7. The gating pore current in DK/R528H channels is carried primarily by Na+. (A) Steady-state currents elicited by 20 ms–step depolarizations from a holding potential of −100 mV in oocytes expressing DK/R528H channels. Currents were initially recorded in external 96 mM Na+ (closed squares). The bath was exchanged with a 110-mM NMDG solution, and then currents were recorded again (open circles). Finally, the external pH was decreased to 6.5, and the pulse protocol was repeated a third time (open triangles). Symbols show means with SEM error bars; n = 5 oocytes. (B) Steady-state current recorded in external Na+ with a symmetrical pH gradient of 7.0 (closed squares), and then repeated after the lower chamber bathing the permeabilized segment of the oocyte was replaced by Mes-buffered solution with pH 5.0. Symbols show mean for DK/WT- (black, n = 3) and DK/R528H- (red, n = 6) expressing oocytes.
	Figure 8. The DK/R528G gating pore is permeable to guanidinium. (A) Currents were recorded from DK/R528G channels in 110 mM NMDG and then in 60 mM NMDG plus 60 mM guanidinium. Traces show responses elicited by step depolarizations of −120 to 10 mV in 5-mV increments from a holding potential of −100 mV. No leak subtraction was used. (B) The steady-state current recorded in NMDG was subtracted from the current in guanidinium plus NMDG and plotted as a function of test potential. Symbols show mean values for oocytes expressing DK/WT (n = 5), DK/R528H (n = 4), or DK/R528G (n = 5) channels. Error bars represent ±SEM.

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