Channel β2–4 subunits fail to substitute for β1 in sensitizing BK channels to lithocholate

https://doi.org/10.1016/j.bbrc.2009.10.091Get rights and content

Abstract

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK α (slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary β subunits (encoded by KCNMB1–4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate β subunit-containing BK channels, yet these steroids appear to be sensed by different types of β subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that β1 sensitizes the BK channel to μM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits (“cbv1”) with β1, β2, β3 or β4 in Xenopus laevis oocytes we now demonstrate that BK β2, β3 and β4 subunits fail to substitute for β1 in providing LC-sensitivity (150 μM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK β1 is highly expressed, while failing to do so in skeletal (flexor digitorum brevis) muscle, where BK β1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by β1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional β1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.

Section snippets

Materials and methods

cRNA preparation and injection into Xenopus laevis oocytes. Full-length cDNA coding for cbv1 subunits (NCBI ID: AY330293) was cloned from freshly isolated rat cerebral artery myocytes as described [17], [18]. Cbv1 cDNA was cleaved from the cloning vector by BamHI (Invitrogen) and XhoI (Promega), and directly inserted into pOX for expression in Xenopus oocytes. pOX-cbv1 was linearized with NotI (Promega) and transcribed in vitro using T3 polymerase. BK β1 subunit cDNA inserted into the

Results and discussion

In order to study the effect of LC on recombinant BK channels of variant subunit composition, we first set to determine the phenotype of BK-mediated currents following injection of oocytes with BK channel-forming (cbv1) subunits with or without BK accessory (β1, β2, β3, or β4) proteins. Macroscopic currents were evoked from I/O patches by 200 ms-long, 10 mV depolarization steps from −150 to 150 mV (Vhold = 0 mV), with Ca2+free = 10 μM (Fig. 1A). As previously shown with other slo1 channels expressed in

Conclusion

Lithocholic acid-induced BK channels activation is conferred by the smooth muscle-abundant β1 subunit but not other β(2–4) subunits. Thus, LC and structural analogs represent a unique tool to probe the presence of functional β1-subunits and activate BK channel in tissues that highly express KCNMB1.

Acknowledgments

We deeply thank Maria Asuncion-Chin (UTHSC) and Aster Sigel (UCLA) for excellent technical assistance. This publication was made possible by NIH Grants R01 HL077424 (A.M.D.) and R01 HL054970 (L.T.). Its contents are solely the responsibility of the authors and do not necessary represent the official views of the NHLBI.

References (39)

  • A. Bukiya et al.

    Structural determinants of monohydroxylated bile acids to activate beta 1 subunit-containing BK channels

    J. Lipid. Res.

    (2008)
  • H. Wang et al.

    Ca2+/calmodulin regulates trafficking of Ca(V)1.2 Ca2+ channels in cultured hippocampal neurons

    J. Neurosci.

    (2007)
  • P. Orio et al.

    New disguises for an old channel: MaxiK channel β-subunits

    News Physiol. Sci.

    (2002)
  • S. Schmid et al.

    To gate or not to gate: are the delta subunits in the glutamate receptor family functional ion channels?

    Mol. Neurobiol.

    (2008)
  • M. Thomsen et al.

    Accessory subunit KChIP2 modulates the cardiac L-type calcium current

    Circ. Res.

    (2009)
  • A. Kato et al.

    Pharmacological regulation of ion channels by auxiliary subunits

    Curr. Opin. Drug Discov. Devel.

    (2007)
  • T. Weiger et al.

    Modulation of calcium-activated potassium channels

    J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol.

    (2002)
  • R. Lu et al.

    MaxiK channel partners: physiological impact

    J. Physiol.

    (2006)
  • L. Salkoff et al.

    High-conductance potassium channels of the SLO family

    Nat. Rev. Neurosci.

    (2006)
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