Figure 1.

FHM mutations increase Ba²⁺ influx through single human Ca_V2.1 channels. (A Top Left) Whole-cell recordings with 5 mM Ba²⁺ as charge carrier. Whole-cell current densities during voltage ramps were measured in HEK293 cells expressing human wt and mutant V1457L (VL) and T666M (TM) Ca_V2.1 channels. Maximal current densities in cells transfected with α_1A-2 and α_1A-2 (-VEA) cDNAs were 70.6 ± 12.0 pA/pF, n = 52, and 71.6 ± 11.3 pA/pF, n = 31, respectively. Identical normalized current density-voltage (I-V) curves were measured for the two wt isoforms (n = 9 and n = 12, respectively). Maximal current densities of the α_1A-2TM and α_1A-2 (-VEA) VL mutants were 10.7 ± 0.9 pA/pF (n = 53) and 22.7 ± 3.1 pA/pF (n = 32), respectively. The I-V curves of the mutants were divided by the maximal current density of the corresponding wt channels. (A Bottom Left) Single-channel cell-attached recordings on HEK cells expressing wt and mutant VL channels; 90 mM Ba²⁺ as charge carrier. Holding potential = −80 mV. Representative current traces from single-channel patches containing a wt Ca_V2.1 channel (cell X24A) or a VL mutant (cell A07B). (Bars = 0.5 pA, 40 ms.) (A Right) Unitary current, i, and open probability, po, as a function of voltage, for wt and VL mutant channels. (Top) Average I-V curves from 19 (wt) and 11 (VL) patches. (Inset) Unitary activity at 20 mV on an expanded time scale. (Bars = 0.3 pA, 10 ms.) (Bottom) Average po-V curves from 12 (wt) and 10 (VL) patches containing a single channel. The data points were best fitted by Boltzmann distributions with V_1/2 = 34.5 mV (k = 6.2) for wt and V_1/2 = 27.4 mV (k = 7.2) for VL. (B) Single-channel cell-attached recordings (90 mM Ba²⁺) on HEK cells expressing wt and mutant TM, VL, R192Q (RQ), V714A (VA), and I1815L (IL) Ca_V2.1 channels. (Top) Product of i and po, as a function of voltage, for the wt channel and the five FHM mutants. All ipo values were divided by the maximal ipo value of the wt channel (−0.21 ± 0.01 pA). Values of i and po for RQ, VA, and IL mutants, and i for TM, were taken from ref. 18; po values for TM were obtained as described in the text. For the wt channel, the normalized ipo values were well fitted by the normalized whole-cell I-V curve, shifted by 26 mV toward more positive voltages (thick line). This shift accounts for the difference in surface potential in the single-channel and whole-cell recording solutions (90 vs. 5 mM Ba²⁺). (Bottom) Comparison of ipo values for the VL mutant, calculated by using either the po values measured in cell-attached recordings (○) or the po values derived from the wt po-V curve in Fig. 1A, shifted in the hyperpolarizing direction of 7.1 mV, corresponding to the difference in V_1/2 activation estimated from fitting the whole-cell current densities of wt and VL channels with Eq. 1 (▴).