Both Transmembrane Domains of BK β1 Subunits Are Essential to Confer the Normal Phenotype of β1-Containing BK Channels

Abstract

Voltage/Ca2+ i-gated, large conductance K+ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel's apparent Ca2+ i sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions (“background”) from β4 showed a phenotype (Vhalf, τact, τdeact) identical to that of complexes containing wt β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β “halves” (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs via the EC loop is also necessary for proper channel function.

Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

“…Their likely interposed location between VSDs of adjacent BKα subunits suggests a role in modulating voltage-sensor activation. The two TM segments in β1 subunit were functionally replaceable by those from the β2 subunit (Orio et al, 2006) but not from the β4 subunit even replaced individually (Kuntamallappanavar, Toro, & Dopico, 2014). The latter showed that both TM segments are required to maintain the characteristic modulatory function of the β1 subunit (Kuntamallappanavar et al, 2014).…”

Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat

“…Furthermore, the parallel leftward shift in the NP o /NP o max -V curve seen at physiological Ca 2+ i is consistent with an increased presence of functional BK β1 subunits [5,11,27,50,51,67,70]. Interestingly, the actual BK V half values in myocytes from BA and CA are similar to those found using heterologous expression systems in which overexpression of β1 subunits is considered to saturate their slo1 partners, including slo1 cloned from rat cerebral artery myocytes ([5,11,27,50,51,70]; e.g., at physiological 3 μM Ca 2+ i , V half values for BA, CA, and slo1+β1 overexpressed in oocytes are 23.5±5.9, 25.15±3.4 and 24.7 ± 13.0 [27]), respectively. Collectively, our electrophysiological data strongly suggest that the increased activity of myocyte BK channels in CA and BA is primarily due to an increased functional presence of BK β1 in these arteries.…”

“…On the other hand, it is widely accepted that within physiological levels of Ca 2+ i and voltage, over-expression of β1 subunits with slo1 in heterologous systems (HEK293 cells, X. laevis oocytes) leads to increased BK steady-state activity [5,11,27,50,51]. The stoichiometry of slo1 and β1 subunits in native channels across different vascular and nonvascular tissues remains largely unknown.…”

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

“…The proximity between the TMs of β1 and the VSD of BK channels makes it tempting to suggest that the TMs of the β1-subunit are involved in the modulation of the voltage-sensor equilibrium between resting and active configurations. This hypothesis is supported by the results of Kuntamallappanavar et al (55), who, by making chimeras between β1 and β4, proposed that both transmembrane segments of β1 are needed to confer the phenotype of α/β1 BK channels. However, the high degree of homology among β-subunit TMs and the differences in the modulation exerted by the different β-subunits (39) cast doubt on the thought that TM segments of β-subunits are the only molecular determinants involved in VSD modulation.…”