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. 2024 Dec;18(1):2396346.
doi: 10.1080/19336950.2024.2396346. Epub 2024 Sep 1.

Evaluation of four KCNMA1 channelopathy variants on BK channel current under CaV1.2 activation

Ria L Dinsdale  1 , Andrea L Meredith  1
Affiliations

Evaluation of four KCNMA1 channelopathy variants on BK channel current under CaV1.2 activation

Ria L Dinsdale et al. Channels (Austin). 2024 Dec.

Abstract

Variants in KCNMA1, encoding the voltage- and calcium-activated K+ (BK) channel, are associated with human neurological disease. The effects of gain-of-function (GOF) and loss-of-function (LOF) variants have been predominantly studied on BK channel currents evoked under steady-state voltage and Ca2+ conditions. However, in their physiological context, BK channels exist in partnership with voltage-gated Ca2+ channels and respond to dynamic changes in intracellular Ca2+ (Ca2+i). In this study, an L-type voltage-gated Ca2+ channel present in the brain, CaV1.2, was co-expressed with wild type and mutant BK channels containing GOF (D434G, N999S) and LOF (H444Q, D965V) patient-associated variants in HEK-293T cells. Whole-cell BK currents were recorded under CaV1.2 activation using buffering conditions that restrict Ca2+i to nano- or micro-domains. Both conditions permitted wild type BK current activation in response to CaV1.2 Ca2+ influx, but differences in behavior between wild type and mutant BK channels were reduced compared to prior studies in clamped Ca2+i. Only the N999S mutation produced an increase in BK current in both micro- and nano-domains using square voltage commands and was also detectable in BK current evoked by a neuronal action potential within a microdomain. These data corroborate the GOF effect of N999S on BK channel activity under dynamic voltage and Ca2+ stimuli, consistent with its pathogenicity in neurological disease. However, the patient-associated mutations D434G, H444Q, and D965V did not exhibit significant effects on BK current under CaV1.2-mediated Ca2+ influx, in contrast with prior steady-state protocols. These results demonstrate a differential potential for KCNMA1 variant pathogenicity compared under diverse voltage and Ca2+ conditions.

Keywords: CACNA1C; CaV1.2; KCa1.1; calcium-activated potassium channel; channelopathy; voltage-gated calcium channels.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Panel A, A cartoon of the subunits comprising the CaV1.2 channel (α1 (CACNA1C), α2β1, and β1b) with the BK channel in micro- and nano-domains. Panel B, A graphical overview of the voltage protocol used to activate channel currents. Panel C, A graphical trace showing total calcium and potassium currents over time. Panel D, A graphical trace showing only the calcium current over time. Panel E, A graphical trace showing only the potassium current over time.
Figure 1.
BK channel activation by Ca2+ influx through CaV1.2 channels. (a) CaV1.2 and BK channel subunits in 2 mM BAPTA and 10 mM EGTA delimited buffering domains. (b) Two-step voltage protocol used to elicit whole-cell CaV1.2 currents (conditioning step), followed by BK currents (test step). (c) Total current from HEK-293T cells co-expressing CaV1.2 and BK channels recorded in 10 mM EGTA. (d) Inward CaV1.2 current isolated by addition of 100 nM paxilline to block BK current. (e) Outward BK channel currents obtained by subtracting (d) from (c). Dotted line represents the zero current level.
Panel A, A series of five graphs showing calcium and potassium current densities across voltages from -100 to +60 mV in EGTA. Panel B, A graph showing the individual values, mean, standard error, and statistically significant conditions of the calcium and potassium currents for each set of co-expressed channels in EGTA. Panel C, A graph showing the individual values, mean, standard error, and statistically significant conditions of the potassium current normalized by the calcium current for each set of co-expressed channels in EGTA.
Figure 2.
CaV1.2 and BK channel currents from cells co-expressing BKWT, BKD434G, BKN999S, BKH444Q and BKD965V in 10 mM EGTA. (a) Current versus conditioning step voltage relationships for CaV1.2 and BKWT (N = 17), BKD434G (N = 13), BKN999S (N = 12), BKH444Q (N = 17) and BKD965V (N = 12) channel currents plotted as a function of the first voltage step of the protocol which elicits Ca2+ influx. Representative traces are displayed in Supplemental Figure 1. (b) Peak CaV1.2 and BK channel current levels from (a). BKN999S currents were larger (p = 0.0034), and BKD965V currents were smaller (p = 0.0122), than BKWT. Expanded y-axis view of BK current levels shown in Supplemental Figure 3. CaV1.2 (BKN999S) currents were reduced compared to CaV1.2 (BKWT; p = 0.0003). (c) Normalized current ratios (IBK/ICav) were increased for CaV1.2 (BKD434G; p = 0.0572) and CaV1.2 (BKN999S; p < 0.0001) compared to CaV1.2 (BKWT). BKWT data on the right-hand side of the split x-axis is replotted for ease of comparison to BKN999S. in B-C panels, values are plotted as individual measurements with average and s.e.m. p values < 0.05 were considered significant.
Panel A, A series of five graphs showing calcium and potassium current densities across voltages from -100 to +60 mV in BAPTA. Panel B, A graph showing the individual values, mean, standard error, and statistically significant conditions of the calcium and potassium currents for each set of co-expressed channels in BAPTA. Panel C, A graph showing the individual values, mean, standard error, and statistically significant conditions of the potassium current normalized by the calcium current for each set of co-expressed channels in BAPTA.
Figure 3.
CaV1.2 and BK channel currents from cells co-expressing BKWT, BKD434G, BKN999S, BKH444Q and BKD965V in 2 mM BAPTA. (A) Current versus conditioning step voltage relationships for CaV1.2 and BKWT (N = 7), BKD434G (N = 10), BKN999S (N = 7), BKH444Q (N = 4) and BKD965V (N = 7) channel currents plotted as a function of the first voltage step of the protocol which elicits Ca2+ influx. Representative traces are displayed in Supplemental Figure 2. (B) Peak CaV1.2 and BK channel current levels from (A). BKN999S currents were larger than BKWT (p = 0.0247), and CaV1.2 (BKD965V) currents were increased compared to CaV1.2 (BKWT)(p = 0.0099). (C) Normalized current ratios (IBK/ICav) were increased for CaV1.2 (BKN999S; p = 0.0319) compared to CaV1.2 (BKWT).
Figure 4.
Figure 4.
Action potential evoked CaV1.2 and BK channel currents from cells co-expressing BKWT, BKD434G, BKN999S, BKH444Q and BKD965V. (A, D) Representative whole-cell CaV1.2 (inward) and BK (outward) currents from cells co-expressing CaV1.2 (BKWT), CaV1.2 (BKD434G), CaV1.2 (BKN999S), CaV1.2 (BKH444Q), and CaV1.2 (BKD965V) channels in 10 mM EGTA (A) and 2 mM BAPTA (D). Traces are normalized to the absolute value of the peak CaV1.2 channel current. Dotted line represents zero current level. Insets: action potential voltage command. (B, E) BK and CaV1.2 channel currents from the peak of the action potential in 10 mM EGTA (B; N = 10-16 per condition) and 2 mM BAPTA (E; N = 17-18 per condition). BKN999S current was larger than BKWT in EGTA (p = 0.0033). BKH444Q current was smaller than BKWT in BAPTA (p = 0.0422). Expanded y-axis view of BK current levels in panel B shown in supplemental Figure 4. No significant differences in CaV1.2 currents were observed. (C, F) normalized current ratios (IBK/ICav) in 10 mM EGTA (C) and 2 mM BAPTA (F). CaV1.2 (BKN999S) channel current was larger compared to CaV1.2 (BKWT) in EGTA (p = 0.003) but not statistically significant in BAPTA (p = 0.0643).
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