PMC9397541_01
PMC9397541_02
Clinical genetics. (A) Pedigree diagram for KCNA1-L155P (red) in the family under study. (B) Sequence alignment for S1 and S1-proximal N-terminal segment of KCNA1 with L155 highlighted (red). (C) Location of L155P and some previously reported EA1 sequence variants in KCNA1 (see Discussion).
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F1
KCNA1, while the proband's father does not harbor the variant. The mutation is apparently de novo in the proband's mother, as the maternal grandmother and grandfather of the proband were tested and were negative (Figure 1A). KCNA1-L155 lies in the S1-proximal portion of the cytoplasmic N-terminus, is highly conserved (Figure 1B), and is close to other EA1-linked sequence variants (Figure 1C). Currents generated by equal co-injection of wild-type and L155P KCNA1 (A1/A1-L155P) generated currents with 39% of the peak current magnitude of wild-type KCNA1 (Figures 2A,B, Table 1). KCNA1 channels, A1/A1-L155P channels exhibited twofold faster activation (Figures 3A,B) and several-fold faster deactivation (depending on voltage) (Figures 3C,D). KCNA1 channels also exhibit voltage-dependent inactivation following activation; compared to wild-type, A1/A1-L155P exhibited negative-shifted voltage dependence of inactivation such that there was, e.g., a threefold greater proportion of A1/A1-L155P channels inactivated at -40 mV compared to wild-type KCNA1, and a -8.4 mV shift in the voltage dependence of inactivation (Figures 3E,F, Table 1).
Effects of L155P on KCNA1 activity. Error bars indicate SEM. n indicates number of oocytes. Statistical comparisons by one-way ANOVA; *P < 0.05; ****P < 0.0001. Dashed line indicates zero current level. (A) Mean traces for wild-type (A1), homozygous mutant (A1-L155P) and heterozygous mutant (A1/A1-L155P) channels expressed in oocytes. Scale bars lower left inset; voltage protocol upper inset; n = 15-29 per group (see Table 1 for values). (B) Mean peak current for traces as in (A); n = 15-29 per group (see Table 1). (C) Mean unclamped oocyte membrane potential for oocytes as in (A); n = 15-29 per group (see Table 1 for values). (D) Inset showing tail currents at -50 mV after prepulses similar to those shown in (A). (E) Mean peak tail current (left) and normalized tail current (right) for currents as in (D) (n = 26-29) (see Table 1 for values).
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Currents generated by equal co-injection of wild-type and L155P KCNA1 (A1/A1-L155P) generated currents with 39% of the peak current magnitude of wild-type KCNA1 (Figures 2A,B, Table 1). Table 1). The shift in membrane potential for oocytes expressing A1/A1-L155P channels was attributable to voltage-independent reduction in current magnitude; there was no quantifiable difference in the voltage dependence of A1/A1-L155P compared to wild-type KCNA1 (Figures 2D,E).
Effects of L155P on KCNA1 gating. Error bars indicate SEM. n indicates number of oocytes. Dashed line indicates zero current level. (A) Mean traces for wild-type (A1) and heterozygous mutant (A1/A1-L155P) channels expressed in oocytes using the voltage protocol shown (upper inset). Scale bars lower left inset; n = 12-16 per group. (B) Mean activation rate (TACT) calculated using a single exponential function from traces as in (A); n = 12-16 per group. (C) Mean traces for wild-type (A1) and heterozygous mutant (A1/A1-L155P) channels expressed in oocytes using the voltage protocol shown (upper inset). Scale bars lower left inset; n = 12-16 per group. (D) Mean deactivation rate (TDeact) calculated using a single exponential function from the circled portion of traces as in (C); n = 12-16 per group. (E) Mean traces for wild-type (A1) and heterozygous mutant (A1/A1-L155P) channels expressed in oocytes using the voltage protocol shown (center inset). Scale bars lower left inset; n = 12-16 per group. (F) Mean proportion of remaining non-inactivated current calculated from the circled portion of traces as in (E); n = 12-16 per group (see Table 1 for values).
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F3
KCNA1 channels, A1/A1-L155P channels exhibited twofold faster activation (Figures 3A,B) and several-fold faster deactivation (depending on voltage) (Figures 3C,D). KCNA1 channels also exhibit voltage-dependent inactivation following activation; compared to wild-type, A1/A1-L155P exhibited negative-shifted voltage dependence of inactivation such that there was, e.g., a threefold greater proportion of A1/A1-L155P channels inactivated at -40 mV compared to wild-type KCNA1, and a -8.4 mV shift in the voltage dependence of inactivation (Figures 3E,F, Table 1).