8.A.14 The Ca²⁺-activated K⁺ Channel auxiliary subunit Slowpoke-β (Sloβ) Family The Sloβ (slowpoke, subunit β) family is a relatively small family of vertebrate homologues. The principal subunit (α) is the large conductance Ca²⁺-activated BK K⁺ channel (TC #1.A.1.3.1), which requires both Ca²⁺ and voltage for opening. Sloβ possesses 2 TMSs in its N- and C-termini, bearing phosphorylation sites in the cytoplasm. The extracellular loop is glycosylated. The Sloβ subunit regulates sensitivity to voltage and Ca²⁺. It may enhance Ca²⁺ sensitivity by altering the conformation and movements of the voltage sensor. A similar function of the beta2 subunit may be governed by a distinct mechanism (Yang et al., 2008). As noted above, the sea anemone neurotoxins modulate sodium channels, and the structures and functional activities have been reviewed (Monastyrnaya et al. 2022). Regulation of voltage-activated K⁺ channel gating by transmembrane β-subunits has been reviewed (Sun et al., 2012). The beta2 subunit of BKCa modulates the apparent Ca²⁺/voltage sensitivity as well as the pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Thus, the beta2 subunit of BKCa channels facilitates channel activation by changing the voltage sensor equilibrium, and this is followed by beta(2)-induced inactivation (Savalli et al. 2007). Coded by a single gene (Slo1, KCM) BK channels are activated by depolarizing potentials and by a rise in intracellular Ca²⁺ concentration. They are large conductance voltage- and Ca²⁺-activated K⁺ channel tetramers characterized by a pore-forming alpha subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K⁺ channels) and a large C-terminus composed of two K⁺ conductance regulatory domains (RCK domains), where the Ca²⁺-binding sites reside. BK channels are associated with accessory beta subunits, and four beta subunits are known (beta1, beta2, beta3, and beta4). Despite the fact that they all share the same topology, each beta subunit has a specific tissue distribution, modifies channel kinetics distinctively, exhibits different pharmacological properties and has different Ca²⁺ sensitivities (Torres et al. 2014). Beta1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17beta-estradiol (E2) increases the BK channel open probability (Po) in SMCs, through a beta1 subunit- dependent modulatory effect. Granados et al. 2019 identified a cluster of hydrophobic residues in the second TMS of beta1, including the residues W163 and F166, as the binding site for E2. The increase in Po induced by E2 is associated with stabilization of the voltage sensor in its active configuration and an increase in the coupling between voltage sensor activation and pore opening (Granados et al. 2019). The transport reaction catalyzed by the BK channel αβ complex is: K⁺ (in) K⁺ (out)
References:
Anjard, C. and W.F. Loomis. (2006). GABA induces terminal differentiation of Dictyostelium through a GABAB receptor. Development 133: 2253-2261.
Behrens, R., A. Nolting, F. Reimann, M. Schwarz, R. Waldschütz, and O. Pongs. (2000). hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel beta subunit family. FEBS Lett. 474: 99-106.
Brenner, R., T.J. Jegla, A. Wickenden, Y. Liu, and R.W. Aldrich. (2000). Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem. 275: 6453-6461.
Bukiya, A.N., A.K. Singh, A.L. Parrill, and A.M. Dopico. (2011). The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory β1 subunit. Proc. Natl. Acad. Sci. USA 108: 20207-20212.
Coetzee, W.A., Y. Amalillo, J. Chiu, A. Chow, D. Lau, T. McCormack, H. Moreno, M.S. Nadal, A. Ozaita, D. Pountney, M. Saganich, E. Vega-Saenz de Miera, and B. Rudy (1999). Molecular diversity of K⁺ channels. Ann. N.Y. Acad. Sci USA 868: 233-285.
Granados, S.T., K. Castillo, F. Bravo-Moraga, R.V. Sepúlveda, W. Carrasquel-Ursulaez, M. Rojas, E. Carmona, Y. Lorenzo-Ceballos, F. González-Nilo, C. González, R. Latorre, and Y.P. Torres. (2019). The molecular nature of the 17β-Estradiol binding site in the voltage- and Ca-activated K (BK) channel β1 subunit. Sci Rep 9: 9965.
Ha, T.S., M.S. Heo, and C.S. Park. (2004). Functional effects of auxiliary beta4-subunit on rat large-conductance Ca²⁺-activated K⁺ channel. Biophys. J. 86: 2871-2882.
Hoshi, T., Y. Tian, R. Xu, S.H. Heinemann, and S. Hou. (2013). Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA. Proc. Natl. Acad. Sci. USA 110: 4822-4827.
Kuntamallappanavar, G. and A.M. Dopico. (2017). BK β1 Subunit-Dependent Facilitation Of Ethanol Inhibition Of BK Current And Cerebral Artery Constriction Is Mediated By The β1 Transmembrane Domain 2. Br J Pharmacol. [Epub: Ahead of Print]
Mangubat, E.Z., T.-T. Tseng, and E. Jakobsson. (2003). Phylogenetic analyses of potassium channel auxiliary subunits. J. Mol. Microbiol. Biotechnol. (in press).
Meera, P., M. Wallner, and L. Toro. (2000). A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca²⁺-activated K⁺ channel resistant to charybdotoxin and iberiotoxin. Proc. Natl. Acad. Sci. USA 97: 5562-5567.
Monastyrnaya, M.M., R.S. Kalina, and E.P. Kozlovskaya. (2022). The Sea Anemone Neurotoxins Modulating Sodium Channels: An Insight at Structure and Functional Activity after Four Decades of Investigation. Toxins (Basel) 15:.
Prabhu, Y., R. Müller, C. Anjard, and A.A. Noegel. (2007). GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development. BMC Dev Biol 7: 44.
Savalli, N., A. Kondratiev, S.B. de Quintana, L. Toro, and R. Olcese. (2007). Modes of operation of the BKCa channel beta2 subunit. J Gen Physiol 130: 117-131.
Sun, X., M.A. Zaydman, and J. Cui. (2012). Regulation of Voltage-Activated K⁺ Channel Gating by Transmembrane β Subunits. Front Pharmacol 3: 63.
Tao, X. and R. MacKinnon. (2019). Molecular structures of the human Slo1 K channel in complex with β4. Elife 8:.
Tseng-Crank, J., N. Godinot, T.E. Johansen, P.K. Ahring, D. Strøbaek, R. Mertz, C.D. Foster, S.P. Olesen, and P.H. Reinhart. (1996). Cloning, expression, and distribution of a Ca²⁺-activated K⁺ channel β-subunit from human brain. Proc. Natl. Acad. Sci. U.S.A. 93: 9200-9205.
Wallner, M., P. Meera, and L. Toro. (1999). Molecular basis of fast inactivation in voltage and Ca²⁺-activated K⁺ channels: a transmembrane beta-subunit homolog. Proc. Natl. Acad. Sci. U.S.A. 96: 4137-4142.
Yang, H., G. Zhang, J. Shi, U.S. Lee, K. Delaloye, and J. Cui. (2008). Subunit-specific effect of the voltage sensor domain on Ca²⁺ sensitivity of BK channels. Biophys. J. 94: 4678-4687.
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Examples:
TC#	Name	Organismal Type	Example
8.A.14.1.1	Sloβ auxiliary subunit; also called KCMNB4, of 210 aas and 2 TMSs, at the N- and C-termini (Behrens et al. 2000). It is 94% identical to the human ortholog and 100% identical to the rat ortholog. It is a regulatory subunit of the calcium activated potassium KCNMA1 (maxiK) channel as it modulates the calcium sensitivity and gating kinetics of KCNMA1 (Brenner et al. 2000). It decreases the gating kinetics and calcium sensitivity of the KCNMA1 channel, but with fast deactivation kinetics. It may also decrease KCNMA1 channel openings at low calcium concentrations but increases channel openings at high calcium concentrations (Ha et al. 2004). beta4 also makes the KCNMA1 channel resistant to 100 nM charybdotoxin (CTX) toxin concentrations (Meera et al. 2000). It's 3-d structure in complex with Slo1 shows it forms a tetramer on the periphery of Slo1 (see TC# 1.A.1.3.10; Tao and MacKinnon 2019).	Animals	*Sloβ from Mus musculus* (Q9JIN6)**

8.A.14.1.2	Smooth muscle and brain Ca²⁺-activated K⁺ channel β-subunit, Slo-β (β1); confers increased Ca²⁺ and voltage sensitivity (Tseng-Crank et al., 1996). A steroid interaction site is in transmembrane domain 2 of the potassium (BK) channel accessory β-subunit (Bukiya et al., 2011). A Long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA), found in oily fish, lower blood pressure by activating vascular BK channels made of Slo1 β1 subunits. Neuronal Slo1 β4 channels were just as well activated by DHA as vascular Slo1 β1 channels, but the stimulatory effect of DHA was much smaller in Slo1 β2, Slo1 LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Residues responsible for this response to DHA were identified (Hoshi et al. 2013). BK beta1 TMS2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral artery constriction at physiological Ca²⁺ and voltages (Kuntamallappanavar and Dopico 2017). After moderate or heavy drinking, the ethanol concentration in the blood is 30 - 60 mM.	Animals	*Sloβ1 of Homo sapiens* (Q16558)**

8.A.14.1.3	Auxillary β-subunit (β2) of voltage-dependent and Ca²⁺ sensitive K⁺ channel (MaxiK; 1.A.1.3.2) The increased current reduces excitability (Wallner et al., 1999). 44% identical to TC#8.A.14.1.4). Full=BK channel subunit beta-2; Short=BKbeta2; Short=Hbeta2; AltName: Full=Calcium-activated potassium channel, subfamily M subunit beta-2; AltName: Full=Charybdotoxin receptor subunit beta-2; AltName: Full=Hbeta3; AltName: Full=K(VCA)beta-2; AltName: Full=Maxi K channel subunit beta-2; AltName: Full=Slo-beta-2. Coexpression of KCNMB2 with the human pore-forming alpha subunit of the large conductance voltage and Ca²⁺-activated K⁺ channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons. β2 not only influences hSlo currents but also limits hSlo surface expression levels via an endocytic mechanism (Zarei et al. 2007).	Animals	*β2 of Homo sapiens* (Q9Y691)**

8.A.14.1.4	Auxillary β subunit (β3) of voltage-dependent and Ca²⁺ sensitive K⁺ channel (MaxiK; 1.A.1.3.2) (Brenner et al., 2000). 44% identical to TC# 8.A.14.1.3.	Animals	*β3 of Homo sapiens* (Q9NPA1)**

Examples:
TC#	Name	Organismal Type	Example