The molecular basis of a severe developmental and neurological disorder associated with a de novo G375R variant of the tetrameric BK channel is unknown. Here, we address this question by recording from single BK channels expressed to mimic a G375R mutation heterozygous with a WT allele. Five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid (heterotetrameric) channels assembled from both mutant and WT subunits. All channel types except WT showed a marked gain-of-function in voltage activation and a smaller decrease-of-function in single-channel conductance, with both changes in function becoming more pronounced as the number of mutant subunits per tetrameric channel increased. The net cellular response from the five different types of channels comprising the molecular phenotype was a shift of −120 mV in the voltage required to activate half of the maximal current through BK channels, giving a net gain-of-function. The WT and homotetrameric mutant channels in the molecular phenotype were consistent with genetic codominance as each displayed properties of a channel arising from only one of the two alleles. The three types of hybrid channels in the molecular phenotype were consistent with partial dominance as their properties were intermediate between those of mutant and WT channels. A model in which BK channels randomly assemble from mutant and WT subunits, with each subunit contributing increments of activation and conductance, approximated the molecular phenotype of the heterozygous G375R mutation.

The BK channel (Slo1, KCa1.1) is a large conductance K+ selective channel that is synergistically activated by Ca2+ and voltage (Barrett et al., 1982; Latorre et al., 1982, 2017; McCobb et al., 1995; Rothberg and Magleby, 2000; Horrigan and Aldrich, 2002; Xia et al., 2002; Salkoff et al., 2006; Horrigan, 2012; Geng and Magleby, 2015; Pantazis and Olcese, 2016; Hite et al., 2017; Tao et al., 2017; Zhou et al., 2017; Tao and MacKinnon, 2019; Geng et al., 2020). BK channels are homotetrameric proteins comprised of four large pore-forming (α) subunits >1,200 amino acids, encoded by the KCNMA1 gene (Fig. S1). BK channels are widely expressed in many cell types where they modulate smooth muscle contraction (Brenner et al., 2000), transmitter release (Robitaille et al., 1993), circadian rhythms (Harvey et al., 2020), repetitive firing (Gu et al., 2007; Park et al., 2022), and cellular excitability (Montgomery and Meredith, 2012). Mutations in the KCNMA1 gene that encode the (α) subunit of BK channels are associated with a wide range of diseases, including epilepsy, dyskinesis, autism, multiple congenital abnormalities, developmental delay, intellectual disability, axial hypotonia, ataxia, cerebral and cerebellar atrophy, bone thickening, and tortuosity of arteries (Wang et al., 2009; Yang et al., 2010; Bailey et al., 2019; Liang et al., 2019; Cui, 2021; Miller et al., 2021).

Studies of the pathogenic properties of BK channels associated with diseases have often been incomplete, focusing on the homotetrameric mutant channels. Yet, for a mutation heterozygous with the wild-type (WT) allele, mutant and WT subunits have the potential to assemble into five different stoichiometries for tetrameric channels with likely differences in functional properties (Fig. 1; MacKinnon, 1991; Blaine and Ribera, 1998; Niu and Magleby, 2002; Bergendahl et al., 2019; Backwell and Marsh, 2022).

Here, we show that this is the case for a de novo G375R mutation in the (α) subunit of BK channels associated with the Liang-Wang Syndrome (Liang et al., 2019). Three unrelated children who carried this mutation had a syndromic neurodevelopmental disorder associated with severe developmental delay and polymalformation syndrome (Liang et al., 2019). The G375R mutation, located on the back side of the S6 pore-lining helix of the α subunit (Fig. S1), replaces the hydrogen side chain of glycine with a large arginine side chain that might be expected to distort the subunit structure and gating (Chen et al., 2014) as well as add a positive charge that may alter conductance.

To assess the functional effects of a G375R mutation, we recorded currents from whole cells and excised macropatches of the membrane after injecting a 1:1 mixture of G375R mutant and WT cRNA encoding mutant and WT subunits into Xenopus laevis oocytes to mimic a de novo mutation heterozygous with a WT allele. When compared with WT, the currents from the 1:1 injection were left-shifted more than −120 mV. This large negative shift in activation indicated a pronounced gain-of-function (GOF) mutation at the cellular level causing the channels to open inappropriately at negative membrane voltages. These observations provide a possible explanation for the severity of the disease associated with the heterozygous G375R mutation.

To investigate the molecular basis underlying the cellular response, detailed single-channel recording (Hamill et al., 1981) following the 1:1 injection suggested that five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid (heterotetrameric) channels. All channel types except WT showed a marked GOF in voltage activation and a smaller decrease-of-function (DOF) in single-channel conductance, with both becoming more pronounced as the number of mutant subunits per channel increased. Codominance was observed for the two homotetrameric channels, with homotetrameric WT channels active at the most positive voltage range of channel activity and homotetrameric mutant channels active at the most negative. Partial dominance was observed for the three types of hybrid channels, which were activated at voltages intermediate between those of WT and homotetrameric mutant channels. A model in which BK channels were randomly assembled from mutant and WT subunits, with each subunit contributing increments of activation and conductance, could approximate the molecular phenotype of the heterozygous G375R BK mutation. The possibility that a channelopathy patient with a heterozygous BK channel mutation synthesizes five different types of BK channels in their neurons and other cells, four with aberrant properties, presents a daunting challenge for treatment.

Oocytes were injected with 0.1–18 ng of cRNA and incubated at 18°C for 2–5 d in Barth’s Solution (in mM): 88 NaCl, 1 KCl, 2.4 NaHC03, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 15 mM HEPES, pH 7.6, plus 12 µM tetracycline. Macro- and single-channel currents were recorded from inside-out patches of the membrane (Hamill et al., 1981) excised from oocytes at room temperature (21–24°C). pClamp 9.0 software (Molecular Devices) was used to drive an Axopatch 200B amplifier to collect the currents. For macropatch current recordings, borosilicate pipettes with 0.5–2 MΩ resistance were used. The macrocurrents were filtered at 10 kHz and sampled at 100 kHz. A minus P/4 protocol was used to remove capacitive transients and leak currents. For single-channel recordings, borosilicate pipettes with 8–12 MΩ resistance were used. The single-channel currents were filtered at 5 kHz and sampled at 200 KHz. The pipette (external) solution contained (in mM) 160 KCl, 2 MgCl2, and 5 TES buffer, pH 7.0. The internal membrane surface of the excised patches was perfused by two different solutions. The designated 0 Ca2+ solution had a free Ca2+ <0.01 µM and contained (in mM) 160 KCl, 1 EGTA, 1 HEDTA, and 10 HEPES, pH 7.0. The solution with 300 µM internal Ca2+ contained (in mM) 160 KCl, 0.3 CaCl2, and 10 HEPES, pH 7.0. Procedures to obtain oocytes from Xenopus laevis were approved by the University of Miami Animal Care and Use Committee. Macropatch and single-channel recordings were analyzed with Clampfit 10.7 software (Molecular Devices) and SigmaPlot 12.

For injection of only G375R cRNA into Xenopus oocytes, we found that it was difficult to get giga-ohm seals of sufficient quality for macropatch recordings using the lower resistance electrodes required for such recordings. Hence, macropatch currents are not presented for injection of only G375R cRNA into oocytes. However, it was still possible to obtain high-quality giga-ohm seals for single-channel recording using the higher resistance pipettes required for such recordings. We also found that the viability of the oocytes was greatly reduced following injection of only G375R cRNA, with the oocytes often starting to die by the second or third day after injection, rather than after a week or more, perhaps because a large fraction of the G375R homotetrameric mutant channels would be expected to be open at resting membrane potentials, as will be shown in later sections. These viability problems were not observed for injection of 1:1 mixtures of G375R mutant and WT cRNA, which gave less pronounced negative shifts in activation, or for injections of only WT cRNA.

The open probability (Po) of each single channel analyzed in detail was determined for a range of voltages with Clampfit 10.7 using 50% threshold analysis to measure open and closed interval durations. Po at each voltage was calculated by dividing the total open time by the sum of the open and closed times. The duration of recordings to estimate Po ranged from ∼5 s to ∼3 min, with the time increasing as the Po decreased. For macropatch current recordings, relative conductance was determined from macroscopic tail current amplitudes using the voltage protocols indicated in the figure legends. G/Gmax vs. V (G-V) plots for macrocurrent recordings and Po vs. V (Po-V) plots for single channels were fitted with a Boltzmann function to estimate voltage for half activation (Vh), the voltage required for half-maximal activation, and b, a measure of voltage sensitivity, usingwhere G/Gmax is the ratio of conductance to maximum conductance and b is the slope factor which gives a measure of voltage sensitivity, where b indicates the change in millivolts required to increase G/Gmax (or Po for single channel recording) e-fold at very low G/Gmax (or Po). Note that an increase in b indicates a decrease in slope and voltage sensitivity.

Single-channel conductance was determined at 100 mV by setting horizontal cursor lines by eye to the open and closed current levels of single-channel recordings.

To examine what effect a de novo G375R mutation of the pore-forming subunit of BK channels would have on channel function when coexpressed with WT subunits (Fig. 1), we first compared the whole-cell macroscopic currents flowing through large numbers of WT BK channels expressed following injection of WT cRNA into Xenopus oocytes with those currents expressed following injection of a 1:1 mixture of mutant and WT cRNA to mimic a heterozygous mutation. The injection of a blank without cRNA served as a control. Voltages were stepped from a holding potential of −80 mV to more negative and more positive voltages to reveal the voltage dependent activation of the expressed currents (Fig. 2 A and Fig. S2). Voltage steps to +50 mV were required to appreciably activate WT BK currents generated from the injection of WT cRNA. In contrast, following a 1:1 injection of G375R mutant and WT cRNA, the expressed BK currents were hyperactive, being already active at −140 mV, with depolarization further increasing the response (Fig. 2 B and Fig. S2). The aberrant BK channels from the 1:1 injection that are responsible for these hyperactive currents could be either homotetrameric mutant channels, hybrid channels, or both (Fig. 1).

The negative shift in activation for currents expressed from the 1:1 injection was so pronounced that large numbers of aberrant BK channels were open at voltages near the resting membrane potential of −60 mV. If aberrant currents expressed similarly in neurons, the flux of K+ through opened BK channels would act to drive the membrane potential toward the equilibrium potential for K+, opposing depolarization of the cell and facilitating repolarization, both of which could interfere with normal neuronal function. Thus, at the level of whole-cell recording, the heterozygous G375R mutation gave a pronounced GOF phenotype because much less depolarization was required to activate BK currents. Some of the disease phenotypes associated with this mutation might be due to such GOF behavior. In contrast, Liang et al. (2019) reported a loss-of-function (LOF) for G375R because they observed no BK currents from HEK293T cells following transfection with G375R cDNA. Their conclusion of a functional LOF vs. our conclusion of a functional GOF will be considered in detail in a later section.

To quantify the average negative voltage shift in activation for the BK currents following injection of a 1:1 mixture of G375R and WT cRNA when compared with WT currents following injection of only WT cRNA, we recorded currents from macropatches of the membrane which were excised from oocytes after channel expression. This allowed the composition of the solution at the inner membrane surface to be controlled, which was not the case for the whole-cell recordings in Fig. 2. For a solution at the intracellular membrane surface containing <0.01 μM Ca2+ (0 Ca2+), WT BK currents did not activate appreciably until the membrane potential exceeded 100 mV (black circles), whereas BK currents from the 1:1 injection of G375R mutant and WT cRNA started to activate at negative voltages of −80 mV (Fig. 3 B). The mean Vh was 182 ± 5.4 mV (n = 6) for WT BK currents and 61.5 ± 16 mV (n = 12) for BK currents from the 1:1 injection, giving a mean left shift of −120 mV in voltage activation for the currents following a 1:1 injection when compared with WT (P < 0.0001). The 1:1 injection also decreased the voltage sensitivity of activation compared with WT with a slope of 38.5 ± 1.8 (n = 8) mV per e-fold change compared with 23.2 ± 1.36 mV (n = 6) for WT currents (P < 0.0001).

The G375R-induced negative shift in voltage activation and reduced slope were even greater in the presence of 300 μM Ca2+ (Fig. S3). Thus, the large negative shift in voltage activation observed for whole-cell recordings following a 1:1 injection of mutant and WT cRNA (Figs. 2 and S2) was also observed for macropatch recordings under conditions in which the intracellular solution and Ca2+ were controlled (Figs. 3 and S3).

Whole-cell and macropatch recordings are useful to show the average response of many hundreds to thousands of BK channels, but they provide limited information about the properties of the underlying channels, unless all channels are identical, which is unlikely to be the case for a heterozygous mutation, as shown in Fig. 1. To investigate the channels underlying the negative shifts in BK currents in Figs. 2 and 3 for a 1:1 injection of mutant and WT cRNA to mimic a mutation heterozygous with the WT allele, we first considered the possible channel types and percentages of expression that might be expected for a heterozygous mutation. We then recorded from individual channels expressed following a 1:1 injection of mutant and WT cRNA to see if the expectations were met.

To identify the types of BK channels expressed following a 1:1 injection of mutant and WT cRNA, the Po-V curves of the 33 individual assembled channels in Fig. 4 B were compared with Po-V curves obtained from individual WT and homotetrameric mutant channels. The WT and homotetrameric mutant channels were obtained by single-channel recording after injection of only WT cRNA or only mutant cRNA, respectively. The WT channels had Vh values that spanned a narrow range from +140 to +176 mV (Fig. 4 C, black Po-V curves), and the homotetrameric mutant channels had Vh values that ranged from −272 mV to −38 mV (Fig. 4 C, blue Po-V curves). The homotetrameric mutant channels had a surprisingly wide range of Vh for channels of the presumed identical composition of four mutant subunits, but all were in a far more negative voltage range than WT channels. The reason for such a wide voltage range in Vh for the homotetrameric mutant channels is not known, but perhaps channels with four mutant subunits can assume different conformations with markedly different activation properties, depending on mutant side-chain orientation (Fig. S1).

To facilitate the identification of the types of assembled channels, the Po-V curves of the individual 33 assembled channels from Fig. 4 B were overlaid on plots of the Vh ranges of the Po-V control curves for known WT (gray shading) and homotetrameric mutant (blue shading) channels in Fig. 4 D. One of the 33 assembled channels had a Po-V curve that overlapped with the known WT channel controls (Fig. 4 D), suggesting that this assembled channel was WT with four WT subunits. 4 of the 33 assembled channels had Po-V curves that overlapped with the known homotetrameric mutant channel controls (Fig. 4 D), suggesting that these four assembled channels were homotetrameric mutants comprised of four mutant subunits. The remaining 28 assembled channels had Po-V curves that did not overlap with the known WT or homotetrameric mutant channel controls (Fig. 4 D) but fell in between, suggesting that these 28 assembled channels were all hybrid channels comprised of a mix of mutant and WT subunits (Fig. 1). In this study, assembled channels with Vh values in the ranges of WT, hybrid, and homotetrameric mutant channels will be referred to as WT, hybrid, and homotetrameric mutant channels, respectively, with the understanding that these classifications are based on Vh values.

For the sampled group of 33 assembled channels, ∼3% (1/33) were consistent with WT, ∼85% (28/33) with hybrid, and ∼12% (4/33) with homotetrameric mutant (Fig. 4, B–D). Fig. 1 predicts ∼6% WT, ∼88% hybrid, and ∼6% homotetrameric mutant channels. Thus, for the G375R mutation heterozygous with WT, both experimental and theoretical considerations suggest that most (85–88%) of the expressed assembled channels will be hybrid, with much smaller fractions of homotetrameric mutant and WT channels. All hybrid and homotetrameric mutant channels displayed negative shifts in activation compared with WT, with the greatest negative shifts for the homotetrameric mutant channels. Consequently, both theoretical and experimental considerations suggest that 94–97% of the BK channels arising from a G375R mutation heterozygous with WT would display aberrant negative shifts in activation, even though only 50% of the subunits synthesized in a cell would be mutant.

Analysis in Fig. 4 suggested that assembled channels comprised of 85% hybrid channels, 3% WT, and 12% homotetrameric mutant (Fig. 4). The 85% hybrid channels themselves could consist of three or four different functional types based on both subunit composition and arrangement (Fig. 1). If the functional properties of hybrid channels depend only on subunit stoichiometry, then three functional types would be expected, for one, two, or three mutant subunits replacing an equal number of WT subunits in the heterotetrameric hybrid channels. In addition, if hybrid channels comprising of two mutants and two WT subunits displayed different functional properties for adjacent or diagonal subunit arrangement, then four types of hybrid channels might be expected (Fig. 1). To assess the number of functional types of hybrid channels, a histogram of the Vh values of the 33 assembled channels from Fig. 4 B was plotted in Fig. 5 A as red bars. Histograms of known WT channel controls (black bars) and homotetrameric mutant channel controls (blue bars) from Fig. 4 C are also plotted. The subunit compositions of the homotetrameric mutant and WT channel controls are known and placed above these two types of channels in Fig. 5 A. As expected from Fig. 4 D, four of the assembled channels had Vh values that overlapped with those of homotetrameric mutant channels, suggesting that they were homotetrameric mutant channels; one assembled channel overlapped with WT, suggesting that it was a WT channel; and the remaining 28 assembled channels had Vh values falling in between those of homotetrameric mutant and WT channels, suggesting they were hybrid channels (Fig. 1) whose properties were determined by mixtures of mutant and WT subunits.

The Vh values of the 28 hybrid channels fell into three apparent clusters with mean values of about 1.4, 59, and 96 mV (Fig. 5 A). Three clusters of Vh for hybrid channels would be expected from Fig. 1 if each mutant subunit replacing a WT subunit added an increment of a negative shift in Vh, independent of subunit arrangement. Accordingly, as a working hypothesis, the different subunit combinations for hybrid channels from Fig. 1 have been placed above the three clusters of hybrid channels in Fig. 5 A to obtain a stepwise negative shift in Vh for each additional mutant subunit replacing a WT subunit. Since only three clusters of hybrid channels were observed, instead of four, it was assumed that the hybrid channels with two mutant and two WT subunits in either adjacent or diagonal subunit arrangement had similar Vh values so that they contributed to the same cluster. Small differences in Vh could be obscured by variability in Vh inherent in single-channel data. Fig. 5 A then suggests five different functional types of assembled channels: homotetrameric mutant, three types of hybrid channels, and WT channels, as indicated.

The percentages of expression of the five functional types of assembled channels were then calculated from the number of assembled channels in each cluster in Fig. 5 A and presented in Fig. 5 B. The results are consistent with 3% WT channels, 12% homotetrameric mutant channels, and 85% hybrid channels, where 30% of the hybrid channels had one mutant and three WT subunits, 34% had two mutant and two WT subunits, and 21% had three mutant and one WT subunits. These percentages can be compared with the theoretical values in Fig. 1 calculated for equal production, and a random assembly of subunits where 6% were WT channels, 6% were homotetrameric mutant channels, and 88% were hybrid channels, where 25% of the hybrid channels had one mutant and three WT subunits, 38% had two mutant and two WT channels, and 25% had three mutant and one WT channels. Simulation of 1 million groups of 33 assembled channels, assuming equal production of mutant and WT subunits followed by random assembly into tetrameric channels, indicated that the experimentally observed percentages in Fig. 5 B were not significantly different (Fig. S4) from the theoretical predictions in Fig. 1. A lack of significance does not exclude the possibility that limited amounts of preferential production and assembly of mutant and WT subunits also contributed to the differences between observed and predicted percentages, in addition to the large variability arising from the random assembly of subunits that are characterized in Fig. S4.

Whereas the observation of three apparent clusters of Vh values for the hybrid assembled channels (Fig. 5) is consistent with theoretical predictions (Fig. 1), peaks can occur by chance alone in histograms of binned data of limited sample size (Miller et al., 1978). Consequently, additional experiments would be needed to determine whether Vh values for hybrid channels can consistently be resolved into three peaks, but an observation of such distinct peaks is not required for support of Fig. 1, as variability in Vh among channels of the same type (McManus and Magleby, 1991) might be sufficient to obscure distinct peaks.

In addition to the negative shift in activation induced by replacing WT subunits with G375R mutant subunits (Figs. 2, 3, 4, and 5), replacing WT subunits with mutant subunits also decreased single-channel conductance (Fig. 6, A and B). The mean single-channel conductance of WT channels was 312 ± 4 pS. This decreased to 245 ± 6 pS for hybrid channels and further decreased to 190 ± 14 pS for homotetrameric mutant channels. These decreases were significant (Fig. 6 legend). Hence, single-channel conductance decreased as mutant subunits replaced WT subunits. The decreased single-channel conductance may arise from the larger volume and positive charge of the mutant arginine side chains replacing the single hydrogen atom of the glycine side chains on one or more of the S6 segments lining the conductance pathway of the BK channel (Fig. S1). The added volume of the sidechains could decrease the volume of the inner vestibule and the added positive charge may act to repel K+ from the inner cavity. Both actions can reduce single-channel conductance in BK channels (Brelidze et al., 2003; Geng et al., 2011; Nimigean et al., 2003).

To examine the relationship between single-channel conductance and Vh, the single-channel conductance for each channel was plotted against Vh for the same channel in Fig. 6 C for the indicated channel types. A linear relationship was observed (Fig. 6 C; R = 0.86, P < 0.0001). When taken together, the data in Fig. 6, B and C, are consistent with the idea that replacing a WT subunit with a mutant subunit adds both an increment of negative voltage shift to Vh and a step decrease in single-channel conductance. Thus, at the single-channel level, the heterozygous G375R mutation acts simultaneously as a GOF mutation to shift voltage activation to more negative voltages (Figs. 4 and 5) and as a DOF mutation to decrease single-channel conductance (Fig. 6). At the whole-cell and macropatch level, the increase in currents from the GOF negative shift in activation would dominate the DOF reduction in single-channel conductance, producing large left-shifted currents (Figs. 2, 3, S2, and S3). The DOF in conductance would act to decrease the consequences of the negative shift in activation.

The heterozygous G375R BK channel variant has been associated with a devastating human phenotype that includes malformation syndrome and severe neurological and developmental disorders (Liang et al., 2019). This variant has only appeared in the human population when heterozygous with WT, perhaps because a homozygous G375R genotype may not permit viability because of the extreme GOF phenotype we observed for homotetrameric mutant channels. To gain insight into the pathogenicity of this variant, currents from whole cells, macropatches, and single channels were recorded from BK channels expressed following a 1:1 injection of G375R mutant and WT cRNA to mimic a G375R mutation heterozygous with WT.

Recordings from whole cells and macropatches, both of which contain many hundreds to thousands of channels, indicated that the Vh of the current following a 1:1 injection, was left shifted to more negative potentials by about −120 mV compared with WT currents (Figs. 2, 3, S2, and S3). The aberrant BK channels underlying the negative shifts in activation would lead to a much greater fraction of BK channels being open at negative membrane potentials, including at potentials near the resting potential (Figs. 2, 3, S2, and S3), which would oppose cellular depolarization, altering cellular function.

To explore the underlying molecular basis for the aberrant current activation associated with the heterozygous G375R variant, we used high-resolution single-channel recording to isolate and characterize the BK channels that are assembled and expressed following injection of a 1:1 mixture of G375R mutant and WT cRNA. Theoretical considerations based on equal production and a random assembly of mutant and WT subunits suggest there could be multiple types of assembled channels with different subunit combinations (MacKinnon, 1991; Blaine and Ribera, 1998; Bergendahl et al., 2019; Backwell and Marsh, 2022; Fig. 1). Consistent with this possibility, our analysis suggested that five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid channels of mixed subunits (Fig. 4; and Fig. 5, A and B). The percentages of expression of these five types of functionally assembled channels were not significantly different (Fig. S4) from the theoretical predictions of Fig. 1 based on equal production and a random assembly of subunits. This suggests, within the limits of experimental variability (Fig. S4), that the processes involved in subunit production, assembly, and expression do not distinguish significantly between mutant and WT alleles and subunits, so the phenotypic differences arise at the functional level of individual channels.

The three types of hybrid channels comprising 85% of the expressed assembled channels had properties falling between those of mutant and WT channels that varied with their apparent subunit composition (Figs. 4, 5, and 6). 97% of the assembled channels, all except for the 3% WT, displayed both GOF negative shifts in activation and smaller DOF reductions in single-channel conductance (Figs. 4, 5, and 6). Hence, most of the channels expressed for the heterozygous G375R mutation displayed aberrant properties. The values of Vh and single channel conductance for each of the five types of functional assembled channels could be predicted with a linear incremental model in which each mutant and WT subunit in each of the five types of functional assembled channels acted relatively independently to contribute increments of both Vh and single-channel conductance to the molecular phenotype of the channel (Fig. 5 C and Fig. 6 D; the models are in the figure legends).

A potential mechanism to produce the five functional types of assembled channels for a heterozygous G375R BK channel mutation, then, is equal production and a random assembly of mutant and WT subunits into channels of five different subunit compositions, where the Vh and single-channel conductance of each channel type are determined by independent contributions from each of the four subunits in a channel. Each WT subunit adds increments of 40.5 mV to Vh and 76.5 pS to single-channel conductance, and each mutant subunit adds increments of −16.7 mV to Vh and 46.1 pS to single-channel conductance (Fig. 5 C and Fig. 6). Whereas a linear incremental model could provide reasonable descriptions of the data, further study is likely to reveal added complexity.

We were surprised to find that the mean macroscopic G-V curve following a 1:1 injection of G375R mutant and WT cRNA could also be well described by a single Boltzmann function (Fig. 3) as the G-V curve arose from the sum of currents from five different types of BK channels with markedly different properties (Figs. 4, 5, and 6). Consequently, an observation that a macroscopic G-V curve is well described by a single Boltzmann function does not necessarily exclude the possibility that the macroscopic G-V curve arises instead from multiple types of channels with different properties. The description with a single Boltzmann function may be possible in this case because the percentages of the five types of contributing channels first increase and then decrease (Fig. 1 and Fig. 5 B), helping to fill in and smooth the G-V curve. Paradoxically, the single-Boltzmann shape of the G-V curves for 1:1 injections and transfections (Fig. 3 and Fig. 7) provides additional evidence for the assembly and expression of hybrid channels, as there would be two clearly separable Boltzmann components in the 1:1 G-V curves arising from homotetrameric mutant and WT channels if the G375R and WT subunits did not coassemble to form three types of hybrid channels with intermediate properties to fill in and smooth the G-V curve.

For classification with regard to genetic disease (Backwell and Marsh, 2022), we suggest that the heterozygous G375R mutation be labeled as an assembly-mediated dominant GOF mutation: assembly-mediated to indicate that mutant and WT subunits assemble into multiple types of functional tetrameric BK channels, dominant because assembled channels with one or more mutant subunits display pathogenic properties, and GOF because less depolarization is required to activate BK channels with one or more mutant subunits. The net result is that 94–97% of the expressed channels display aberrant activation (Figs. 1, 4, and 5). A dominant GOF phenotype has been described previously for the heterozygous G88R mutation of the TASK-4 K+ channel in the heart, based on a study using whole-cell currents (Friedrich et al., 2014).

An assembly-mediated dominant-GOF mutation for heterozygous G375R can be compared to the well-known dominant-negative effect observed for some types of protein complexes and channelopathies (Fink et al., 1996; Ribera et al., 1996; Silberberg et al., 2005; Reed et al., 2016; Du et al., 2020; Hichri et al., 2020; Backwell and Marsh, 2022). Both can cause disease through a disproportionate fraction of the channels affected. In the first case, there is a GOF in channels with one or more mutant subunits, and in the second there is a complete or partial loss of function of channels associated with, typically, one or more mutant subunits.

Whereas an assembly-mediated dominant GOF classification of the heterozygous G375R mutation is useful to suggest the potential underlying basis of genetic disease, as it describes the net functional cellular phenotype, the molecular phenotype is more complex. The action of the heterozygous G375R mutation is to generate five types of channels, four of which simultaneously display a GOF in activation and a smaller DOF in single-channel conduction. The GOF dominates the response at the level of cellular currents.

The random assembly of mutant and WT subunits into tetrameric BK channels when mimicking a heterozygous mutation led to multiple types of genetic dominance at the level of the molecular phenotype when viewed with single-channel recording. Codominance was observed for the WT and homotetrameric mutant channels, and partial dominance was observed for the three types of hybrid channels. Support for codominance was that the homotetrameric mutant and WT channels expressed in the molecular phenotype when mimicking a heterozygous mutation had the same molecular phenotypes as homotetrameric mutant and WT channels expressed following injection of only mutant or WT cRNA (Figs. 4, 5, and 6). Support for partial dominance was that the three types of expressed hybrid channels had individual values of Vh and single channel conductance that were distinct from each other and fell in the range between those of homotetrameric mutant and WT channels (Fig. 5 C and Fig. 6 D). The levels of partial dominance in the linear incremental model (Fig. 5 C and Fig. 6 D) were determined by the numbers of mutant and WT subunits per channel acting independently of one another, rather than by mutant subunits altering the function of WT subunits, as in some classical descriptions of genetic dominance. It is remarkable that a single base pair substitution in one allele of a pair of alleles that encode for the α subunit of BK channels results in the expression of WT BK channels plus four aberrant types of BK channels, each with different molecular phenotypes that simultaneously display a dominant GOF in Vh and a lessor DOF in single-channel conductance, with the five types of functional channels displaying either codominance or one of three levels of partial dominance for both Vh and single channel conductance.

How do our observations of independent interactions of the G375R mutant and WT pore-forming subunits compare to interactions of these subunits with regulatory subunits? Wang et al. (2002) found that single BK channels comprised of four subunits could be associated with zero to four regulatory β2-subunits per channel, with each β2-subunit giving incremental changes in Vh. Hence, both mutated and WT pore-forming subunits (Fig. 5 C; Niu and Magleby, 2002) and the non-pore-forming regulatory β2-subunits (Wang et al., 2002) can give incremental changes in gating, but such incremental changes per subunit are not necessarily universal, so each type of subunit will need to be assessed. A single BK channel comprised of four pore-forming subunits can also include up to four regulatory γ1-subunits, but a single γ1-subunit per channel is sufficient to induce the full gating shift induced by γ1-subunits (Gonzalez-Perez et al., 2014, 2018).

Devising therapies for the de novo G375R heterozygous mutation will be challenging. Theoretical and experimental observations (Figs. 1, 4, and 5) suggest that most (94–97%) of the channels for a mutation heterozygous with WT would be pathogenic, with only 3–6% of the channels WT. The pathogenic channels would consist of multiple channel types, each with large differences in activation properties and smaller differences in conductance (Figs. 4, 5, and 6). Therapies to block or inactivate the pathogenic channels would ideally silence the multiple types of pathogenic channels while leaving any WT channels intact. Even if such selective blockers could be devised, it is unlikely that the remaining 3–6% of WT channels would be sufficient to restore normal cellular function. Effective therapies will likely require replacing or silencing the mutant alleles or preventing mutant subunits from assembling with themselves and WT subunits if/when such techniques become practical in humans.