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N-terminal Isoforms of the Large-conductance Ca2+-activated K+ Channel Are Differentially Modulated by the Auxiliary β1-Subunit*

  • Ramón A. Lorca
    Affiliations
    Department of Obstetrics and Gynecology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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  • Susan J. Stamnes
    Affiliations
    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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  • Meghan K. Pillai
    Affiliations
    Department of Obstetrics and Gynecology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110
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  • Jordy J. Hsiao
    Affiliations
    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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  • Michael E. Wright
    Affiliations
    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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  • Sarah K. England
    Correspondence
    To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Washington University in St. Louis School of Medicine, 425 South Euclid Ave., Campus Bx. 8064, St. Louis, MO 63110. Tel.: 314-286-1798; Fax: 314-747-4150
    Affiliations
    Department of Obstetrics and Gynecology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant R01 HD037831 and American Heart Association Grant 12GRNT10990002 (to S. K. E.), American Heart Association Postdoctoral Fellowship 12POST10660000 (to R. A. L.), and National Center for Research Resources, Clinical and Translational Science Award M01-RR-00059 for human tissue obtainment.
Open AccessPublished:February 25, 2014DOI:https://doi.org/10.1074/jbc.M113.521526
      The large-conductance Ca2+-activated K+ (BKCa) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BKCa α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BKCa α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μm and 100 μm Ca2+. The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BKCa channel activity in cells, highlighting a novel mechanism of BKCa channel regulation.

      Introduction

      The large-conductance voltage- and Ca2+-activated K+ channel (BKCa)
      The abbreviation used is: BKCa
      large-conductance voltage- and Ca2+-activated K+ channel.
      is a key regulator of membrane excitability in a wide variety of cells such as neurons, smooth muscle, chromaffin cells, and immune cells (
      • Scott R.S.
      • Bustillo D.
      • Olivos-Oré L.A.
      • Cuchillo-Ibañez I.
      • Barahona M.V.
      • Carbone E.
      • Artalejo A.R.
      Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells.
      ,
      • Yazejian B.
      • Sun X.P.
      • Grinnell A.D.
      Tracking presynaptic Ca2+ dynamics during neurotransmitter release with Ca2+-activated K+ channels.
      ,
      • Essin K.
      • Gollasch M.
      • Rolle S.
      • Weissgerber P.
      • Sausbier M.
      • Bohn E.
      • Autenrieth I.B.
      • Ruth P.
      • Luft F.C.
      • Nauseef W.M.
      • Kettritz R.
      BK channels in innate immune functions of neutrophils and macrophages.
      ,
      • Korovkina V.P.
      • Brainard A.M.
      • England S.K.
      Translocation of an endoproteolytically cleaved maxi-K channel isoform: mechanisms to induce human myometrial cell repolarization.
      • Brayden J.E.
      • Nelson M.T.
      Regulation of arterial tone by activation of calcium-dependent potassium channels.
      ). BKCa channel activation integrates both membrane depolarization and increases in intracellular Ca2+ to control membrane excitability (
      • Cui J.
      • Cox D.H.
      • Aldrich R.W.
      Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels.
      ). Although a single gene (KCNMA1) encodes the BKCa channel α-subunit, alternative splicing produces an array of BKCa isoforms that respond to a variety of modulators in tissue-specific manners (
      • Korovkina V.P.
      • Brainard A.M.
      • England S.K.
      Translocation of an endoproteolytically cleaved maxi-K channel isoform: mechanisms to induce human myometrial cell repolarization.
      ,
      • Tian L.
      • Duncan R.R.
      • Hammond M.S.
      • Coghill L.S.
      • Wen H.
      • Rusinova R.
      • Clark A.G.
      • Levitan I.B.
      • Shipston M.J.
      Alternative splicing switches potassium channel sensitivity to protein phosphorylation.
      ,
      • Chen L.
      • Tian L.
      • MacDonald S.H.
      • McClafferty H.
      • Hammond M.S.
      • Huibant J.M.
      • Ruth P.
      • Knaus H.G.
      • Shipston M.J.
      Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) α-subunits generated from a single site of splicing.
      • Erxleben C.
      • Everhart A.L.
      • Romeo C.
      • Florance H.
      • Bauer M.B.
      • Alcorta D.A.
      • Rossie S.
      • Shipston M.J.
      • Armstrong D.L.
      Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.
      ). Despite the high degree of splicing, all BKCa α-subunits are composed of seven conserved transmembrane domains (S0 through S6) and an extracellular N terminus. The N terminus and first transmembrane domain (S0) of the α-subunit are required for association with the auxiliary β1-subunit (
      • Wallner M.
      • Meera P.
      • Toro L.
      Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
      ,
      • Morrow J.P.
      • Zakharov S.I.
      • Liu G.
      • Yang L.
      • Sok A.J.
      • Marx S.O.
      Defining the BK channel domains required for β1-subunit modulation.
      ), which is an important source of variable regulation for BKCa channel function (
      • Tanaka Y.
      • Meera P.
      • Song M.
      • Knaus H.G.
      • Toro L.
      Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α + β subunit complexes.
      ,
      • McManus O.B.
      • Helms L.M.
      • Pallanck L.
      • Ganetzky B.
      • Swanson R.
      • Leonard R.J.
      Functional role of the β subunit of high conductance calcium-activated potassium channels.
      • Orio P.
      • Torres Y.
      • Rojas P.
      • Carvacho I.
      • Garcia M.L.
      • Toro L.
      • Valverde M.A.
      • Latorre R.
      Structural determinants for functional coupling between the β and α subunits in the Ca2+-activated K+ (BK) channel.
      ).
      Three possible translation initiation sites have been identified in the BKCa α-subunit N-terminal sequence (
      • Wallner M.
      • Meera P.
      • Ottolia M.
      • Kaczorowski G.J.
      • Latorre R.
      • Garcia M.L.
      • Stefani E.
      • Toro L.
      Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
      ,
      • McCobb D.P.
      • Fowler N.L.
      • Featherstone T.
      • Lingle C.J.
      • Saito M.
      • Krause J.E.
      • Salkoff L.
      A human calcium-activated potassium channel gene expressed in vascular smooth muscle.
      ). Heterologous expression studies have used the third initiation site (methionine at position 66), starting with amino acid sequence MDAL, as the canonical human BKCa channel α-subunit to investigate β-subunit modulation (
      • Wallner M.
      • Meera P.
      • Toro L.
      Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
      ,
      • Orio P.
      • Torres Y.
      • Rojas P.
      • Carvacho I.
      • Garcia M.L.
      • Toro L.
      • Valverde M.A.
      • Latorre R.
      Structural determinants for functional coupling between the β and α subunits in the Ca2+-activated K+ (BK) channel.
      ,
      • Contreras G.F.
      • Neely A.
      • Alvarez O.
      • Gonzalez C.
      • Latorre R.
      Modulation of BK channel voltage gating by different auxiliary β subunits.
      ). Other studies of the mouse (
      • McManus O.B.
      • Helms L.M.
      • Pallanck L.
      • Ganetzky B.
      • Swanson R.
      • Leonard R.J.
      Functional role of the β subunit of high conductance calcium-activated potassium channels.
      ,
      • Nimigean C.M.
      • Magleby K.L.
      Functional coupling of the β(1) subunit to the large conductance Ca2+-activated K+ channel in the absence of Ca2+. Increased Ca2+ sensitivity from a Ca2+-independent mechanism.
      ) or rat (
      • Erxleben C.
      • Everhart A.L.
      • Romeo C.
      • Florance H.
      • Bauer M.B.
      • Alcorta D.A.
      • Rossie S.
      • Shipston M.J.
      • Armstrong D.L.
      Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.
      ) BKCa channel have also used N-terminal truncated isoforms that start at the second or third translation initiation sites (starting with MSSN or MDAL, respectively). The full-length transcript, which can encode an isoform that starts with MANG, has been isolated from human smooth muscle (
      • Wallner M.
      • Meera P.
      • Ottolia M.
      • Kaczorowski G.J.
      • Latorre R.
      • Garcia M.L.
      • Stefani E.
      • Toro L.
      Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
      ), but whether the β1-subunit modulates this isoform of the human BKCa α-subunit has not been extensively investigated. Because the N-terminal region and first transmembrane domain (S0) are necessary for β1-subunit modulation (
      • Wallner M.
      • Meera P.
      • Toro L.
      Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
      ,
      • Morrow J.P.
      • Zakharov S.I.
      • Liu G.
      • Yang L.
      • Sok A.J.
      • Marx S.O.
      Defining the BK channel domains required for β1-subunit modulation.
      ), we hypothesized that these three N-terminal isoforms of the BKCa α-subunit may be differentially modulated by the accessory β1-subunit. Here, we report that the β1-subunit differentially modulates the activation of the MANG, MSSN, and MDAL isoforms. We show that β1 modulates the voltage activation of the N-terminally extended isoforms less than that of the MDAL isoform. This effect may reflect differential modulation of channel kinetics of the three isoforms by β1. By contrast, the β2-subunit modulated voltage activation of MDAL, MSSN, and MANG isoforms to similar extents. These results suggest a new mechanism of fine-tuning the BKCa channel activity that depends on the expression of the N-terminal isoforms and their modulation by the β1-subunit.

      DISCUSSION

      The BKCa channel plays an important role in regulating the membrane potential of excitable cells. Association of the pore-forming α-subunit of the BKCa channel with distinct auxiliary subunits (β1–β4 or the recently described γ1–γ4) is a significant form of channel modulation (
      • Contreras G.F.
      • Neely A.
      • Alvarez O.
      • Gonzalez C.
      • Latorre R.
      Modulation of BK channel voltage gating by different auxiliary β subunits.
      ,
      • Yan J.
      • Aldrich R.W.
      BK potassium channel modulation by leucine-rich repeat-containing proteins.
      ). Accordingly, interaction of the α-subunit with the β1-subunit, which predominates in smooth muscle, confers increased Ca2+ sensitivity and decreased voltage-dependence to the BKCa channel (
      • Tanaka Y.
      • Meera P.
      • Song M.
      • Knaus H.G.
      • Toro L.
      Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α + β subunit complexes.
      ,
      • McManus O.B.
      • Helms L.M.
      • Pallanck L.
      • Ganetzky B.
      • Swanson R.
      • Leonard R.J.
      Functional role of the β subunit of high conductance calcium-activated potassium channels.
      ). Both the N-terminal region and the S0 transmembrane domain of the BKCa α-subunit are essential for both its interaction with the auxiliary β1-subunit (
      • Wallner M.
      • Meera P.
      • Toro L.
      Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
      ,
      • Morrow J.P.
      • Zakharov S.I.
      • Liu G.
      • Yang L.
      • Sok A.J.
      • Marx S.O.
      Defining the BK channel domains required for β1-subunit modulation.
      ) and for β2-subunit modulation (
      • Lee U.S.
      • Shi J.
      • Cui J.
      Modulation of BK channel gating by the β2 subunit involves both membrane-spanning and cytoplasmic domains of Slo1.
      ). In this work, we studied the properties of three different N-terminal isoforms (MANG, MSSN, and MDAL) and how they are modulated by the auxiliary β1- and β2-subunits. We observed that whereas the voltage-activation curve of MDAL was shifted to the left by ∼50 mV, that of MANG was only shifted by ∼25 mV, and that of the MSSN isoform was not affected by the β1-subunit.
      The diminished β1-induced shift in voltage-activation curves observed in the MANG isoform, and the lack of modulation in MSSN, were reflected in the modulation of channel kinetics. We observed that the β1-subunit modulated channel kinetics of all three BKCa α-subunit N-terminal isoforms, as evidenced by increases in open dwell times. We also observed that the closed dwell times of MANG and MDAL decreased, whereas the closed dwell times of MSSN increased; these data are consistent with other reports showing that the β1-subunit modifies the kinetics of the channel by stabilizing its open state (
      • Orio P.
      • Torres Y.
      • Rojas P.
      • Carvacho I.
      • Garcia M.L.
      • Toro L.
      • Valverde M.A.
      • Latorre R.
      Structural determinants for functional coupling between the β and α subunits in the Ca2+-activated K+ (BK) channel.
      ,
      • Nimigean C.M.
      • Magleby K.L.
      Functional coupling of the β(1) subunit to the large conductance Ca2+-activated K+ channel in the absence of Ca2+. Increased Ca2+ sensitivity from a Ca2+-independent mechanism.
      ). The β1-subunit also increases the apparent Ca2+ sensitivity of the channel by increasing burst duration (
      • Nimigean C.M.
      • Magleby K.L.
      The β subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states.
      ). Analysis of single-channel kinetics revealed that both MDAL and MSSN isoforms were maintained in the bursting state by β1, as indicated by an increased burst duration, but the burst duration of the MANG isoform was not affected by the β1-subunit. Furthermore, β1 increased the interval between bursts of MANG and MSSN isoforms but did not change the interval of the MDAL isoform. These observations (summarized in Table 3) support a model of allosteric modulation of BKCa channel kinetics by the β1-subunit in which the MANG-extended N-terminal sequence reduces the β1-dependent stabilization of the bursting state, but not the change in overall open and closed dwell times. In the MSSN isoform, the bursting state is stabilized by β1, but the time between bursts is longer, which might explain the net null effect of β1 on the voltage-activation of the MSSN isoform. Differential modifications by β1 in the burst duration and open/closed time constants of these channels could lead to changes in Po and apparent Ca2+ sensitivity (
      • Nimigean C.M.
      • Magleby K.L.
      The β subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states.
      ), altering membrane excitability. Thus, β1-dependent increase in burst duration of the MDAL isoform will result in an increase of the overall time the channel remains in the open state, a greater Po, and a subsequent leftward shift in voltage activation.
      TABLE 3Summary of the β1-subunit effect on the different properties of BKCa channel α-subunit N-terminal isoforms
      IsoformVoltage dependenceConductanceOpen dwell time constantsClose dwell time constantsBurst durationInterburst interval
      MANGLeftward shiftNo changeBoth increasedDecreasedNo changeIncreased
      MSSNNo shiftNo changeOnly τ2 increasedIncreasedIncreasedIncreased
      MDALLarger leftward shiftDecreasedBoth increasedDecreasedIncreasedNo change
      The association between BKCa α- and its β1-subunit plays an important role in multiple tissues, most notably in regulating smooth muscle contractility (
      • Tanaka Y.
      • Meera P.
      • Song M.
      • Knaus H.G.
      • Toro L.
      Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α + β subunit complexes.
      ,
      • Behrens R.
      • Nolting A.
      • Reimann F.
      • Schwarz M.
      • Waldschütz R.
      • Pongs O.
      hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel β subunit family.
      ,
      • Knaus H.G.
      • Folander K.
      • Garcia-Calvo M.
      • Garcia M.L.
      • Kaczorowski G.J.
      • Smith M.
      • Swanson R.
      Primary sequence and immunological characterization of β-subunit of high conductance Ca2+-activated K+ channel from smooth muscle.
      ,
      • Brenner R.
      • Peréz G.J.
      • Bonev A.D.
      • Eckman D.M.
      • Kosek J.C.
      • Wiler S.W.
      • Patterson A.J.
      • Nelson M.T.
      • Aldrich R.W.
      Vasoregulation by the β1 subunit of the calcium-activated potassium channel.
      ,
      • Plüger S.
      • Faulhaber J.
      • Fürstenau M.
      • Löhn M.
      • Waldschütz R.
      • Gollasch M.
      • Haller H.
      • Luft F.C.
      • Ehmke H.
      • Pongs O.
      Mice with disrupted BK channel beta1 subunit gene feature abnormal Ca2+ spark/STOC coupling and elevated blood pressure.
      • Petkov G.V.
      • Bonev A.D.
      • Heppner T.J.
      • Brenner R.
      • Aldrich R.W.
      • Nelson M.T.
      β1-subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle.
      ). For example, the β1-subunit controls arterial tone in resistance arteries (
      • Brenner R.
      • Peréz G.J.
      • Bonev A.D.
      • Eckman D.M.
      • Kosek J.C.
      • Wiler S.W.
      • Patterson A.J.
      • Nelson M.T.
      • Aldrich R.W.
      Vasoregulation by the β1 subunit of the calcium-activated potassium channel.
      ) and contractility in the urinary bladder (
      • Petkov G.V.
      • Bonev A.D.
      • Heppner T.J.
      • Brenner R.
      • Aldrich R.W.
      • Nelson M.T.
      β1-subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle.
      ). Because the interaction between BKCa α and β1 seems to be important for human myometrial contractility (
      • Matharoo-Ball B.
      • Ashford M.L.
      • Arulkumaran S.
      • Khan R.N.
      Down-regulation of the α- and β-subunits of the calcium-activated potassium channel in human myometrium with parturition.
      ,
      • Brainard A.M.
      • Korovkina V.P.
      • England S.K.
      Potassium channels and uterine function.
      ), and both subunits are abundantly expressed in this tissue (
      • Chan Y.W.
      • van den Berg H.A.
      • Moore J.D.
      • Quenby S.
      • Blanks A.M.
      Assessment of myometrial transcriptome changes associated with spontaneous human labour by high throughput RNA-seq.
      ), we explored the presence of the different N-terminal isoforms in smooth muscle tissue derived from human myometrium obtained from non-laboring pregnant women at term. However, detection of peptides corresponding to the various N termini by using mass spectrometry was unsuccessful (data not shown), likely because the low complexity of the sequence between amino acids Met-1 and Met-66 (polyglycine and polyserine stretches, Fig. 1) complicates the detection of these peptides by mass spectrometry. Further studies, such as N-terminal protein sequencing or development of specific antibodies targeted to the N-terminal region of the BKCa α-subunit, will be necessary to determine the endogenous expression of these isoforms.
      Our data suggest that the additional N-terminal sequence in the MANG isoform might dampen the effects of the β1-subunit on channel activation by a specific allosteric mechanism rather than by hampering protein-protein interaction. The case of MSSN seems to be more complex; extending the N-terminal sequence by 41 amino acids completely abolished the modulation by β1 of the voltage-dependent activation observed with MDAL, but adding 24 more amino acids partially restored this modulation, as observed with the MANG isoform. The secondary structure of this N-terminal sequence is not clear because of its low complexity poly-glycine and poly-serine stretches (Fig. 1). Further studies aimed at dissecting the length of residues necessary to prevent and restore β1 modulation might elucidate the structural determinants of this effect.
      Several studies have reported that the β1-subunit increases the Ca2+ and voltage sensitivity of recombinant BKCa channels (
      • Erxleben C.
      • Everhart A.L.
      • Romeo C.
      • Florance H.
      • Bauer M.B.
      • Alcorta D.A.
      • Rossie S.
      • Shipston M.J.
      • Armstrong D.L.
      Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.
      ,
      • Wallner M.
      • Meera P.
      • Toro L.
      Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
      • Morrow J.P.
      • Zakharov S.I.
      • Liu G.
      • Yang L.
      • Sok A.J.
      • Marx S.O.
      Defining the BK channel domains required for β1-subunit modulation.
      ,
      • McManus O.B.
      • Helms L.M.
      • Pallanck L.
      • Ganetzky B.
      • Swanson R.
      • Leonard R.J.
      Functional role of the β subunit of high conductance calcium-activated potassium channels.
      ,
      • Orio P.
      • Torres Y.
      • Rojas P.
      • Carvacho I.
      • Garcia M.L.
      • Toro L.
      • Valverde M.A.
      • Latorre R.
      Structural determinants for functional coupling between the β and α subunits in the Ca2+-activated K+ (BK) channel.
      • Wallner M.
      • Meera P.
      • Ottolia M.
      • Kaczorowski G.J.
      • Latorre R.
      • Garcia M.L.
      • Stefani E.
      • Toro L.
      Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
      ,
      • Contreras G.F.
      • Neely A.
      • Alvarez O.
      • Gonzalez C.
      • Latorre R.
      Modulation of BK channel voltage gating by different auxiliary β subunits.
      ,
      • Nimigean C.M.
      • Magleby K.L.
      Functional coupling of the β(1) subunit to the large conductance Ca2+-activated K+ channel in the absence of Ca2+. Increased Ca2+ sensitivity from a Ca2+-independent mechanism.
      ,
      • Cox D.H.
      • Aldrich R.W.
      Role of the β1 subunit in large-conductance Ca2+-activated K+ channel gating energetics. Mechanisms of enhanced Ca2+ sensitivity.
      ,
      • Bao L.
      • Cox D.H.
      Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its β1 subunit.
      • Sun X.
      • Shi J.
      • Delaloye K.
      • Yang X.
      • Yang H.
      • Zhang G.
      • Cui J.
      The interface between membrane-spanning and cytosolic domains in Ca2+-dependent K+ channels is involved in β subunit modulation of gating.
      ). However, most reports used an α-subunit isoform starting at the third initiation site, the MDAL isoform (
      • Wallner M.
      • Meera P.
      • Toro L.
      Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
      ,
      • Orio P.
      • Torres Y.
      • Rojas P.
      • Carvacho I.
      • Garcia M.L.
      • Toro L.
      • Valverde M.A.
      • Latorre R.
      Structural determinants for functional coupling between the β and α subunits in the Ca2+-activated K+ (BK) channel.
      ,
      • Wallner M.
      • Meera P.
      • Ottolia M.
      • Kaczorowski G.J.
      • Latorre R.
      • Garcia M.L.
      • Stefani E.
      • Toro L.
      Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
      ,
      • Contreras G.F.
      • Neely A.
      • Alvarez O.
      • Gonzalez C.
      • Latorre R.
      Modulation of BK channel voltage gating by different auxiliary β subunits.
      ). Although three predicted initiation codons are encoded in the DNA sequence of the α-subunit (
      • Wallner M.
      • Meera P.
      • Ottolia M.
      • Kaczorowski G.J.
      • Latorre R.
      • Garcia M.L.
      • Stefani E.
      • Toro L.
      Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
      ,
      • McCobb D.P.
      • Fowler N.L.
      • Featherstone T.
      • Lingle C.J.
      • Saito M.
      • Krause J.E.
      • Salkoff L.
      A human calcium-activated potassium channel gene expressed in vascular smooth muscle.
      ), neither the first nor second initiation sites (corresponding to MANG and MSSN isoforms, respectively) generate functional channels when expressed in Xenopus laevis oocytes (
      • Wallner M.
      • Meera P.
      • Ottolia M.
      • Kaczorowski G.J.
      • Latorre R.
      • Garcia M.L.
      • Stefani E.
      • Toro L.
      Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
      ). One study demonstrated that expression of functional BKCa channels might depend on the taxonomic class of the cell line used for heterologous expression. Erxleben et al. (
      • Erxleben C.
      • Everhart A.L.
      • Romeo C.
      • Florance H.
      • Bauer M.B.
      • Alcorta D.A.
      • Rossie S.
      • Shipston M.J.
      • Armstrong D.L.
      Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.
      ) described a BKCa channel in a rat pituitary cell line, starting at MSSN, whose kinetics are slower in the presence of β1, but lack the typical change in Ca2+ sensitivity at 1–10 μm Ca2+. Interestingly, a truncated form starting at MDAL restores modulation by the β1-subunit. These authors propose that the poly-serine stretch between Met-25 and Met-66 is important in blocking Ca2+ sensitivity modulation by β1. We found comparable results in that the human MSSN isoform lacked, and the MDAL isoform retained, voltage-dependent activation modulation by the β1-subunit. In our study, using a mammalian heterologous expression system, both MANG and MSSN N-terminal isoforms formed functional channels with intrinsic properties indistinguishable from those observed in the MDAL isoform. In addition, expression of the N-terminal isoforms in a mouse fibroblast cell line also generated functional channels, comparable to those expressed in HEK293T cells (data not shown). Thus, expression systems may account for the differences in BKCa channel N-terminal α-subunit function observed between our experimental conditions and those in other reports; however, the underlying mechanism for tissue specific expression of these N-terminal isoforms is still unclear.
      The functional complexity of the N-terminal BKCa isoforms, and how their expression is regulated, has not been fully explored. Alternative translation initiation has been described to modulate the expression and function of N-terminal truncated forms of certain potassium channels: the voltage-gated potassium channel Kv3.3 (
      • Fernandez F.R.
      • Morales E.
      • Rashid A.J.
      • Dunn R.J.
      • Turner R.W.
      Inactivation of Kv3.3 potassium channels in heterologous expression systems.
      ) and the two P-domain potassium channels K2P2.1 and K2P10.1 (
      • Simkin D.
      • Cavanaugh E.J.
      • Kim D.
      Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation.
      ,
      • Thomas D.
      • Plant L.D.
      • Wilkens C.M.
      • McCrossan Z.A.
      • Goldstein S.A.
      Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium.
      ). Alternative translation initiation is a mechanism to regulate protein diversity whereby proteins with different N termini are produced from a single mRNA (
      • Cai J.
      • Huang Y.
      • Li F.
      • Li Y.
      Alteration of protein subcellular location and domain formation by alternative translational initiation.
      ). Alternative translation initiation occurs during translation when the ribosome binds to an initiation codon (AUG) that is not the first cap-proximal in the mRNA coding region, thereby generating N-terminally truncated protein isoforms. Generally, AUG sequences are flanked by Kozak consensus sequences, which facilitate binding of the ribosome to the AUG sequence and thus translation initiation (
      • Kozak M.
      Structural features in eukaryotic mRNAs that modulate the initiation of translation.
      ). The extent of this facilitation is determined by the relative strength of the ribosome binding given by the Kozak sequence; thus, some sequences will promote binding of the ribosome to a certain initiation site over others (
      • Kozak M.
      Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6.
      ). In examining the mRNA sequence of the BKCa channel α-subunit, we found that the third initiation codon (Met-66) is flanked by a Kozak sequence stronger than those found in the first or second initiation codon (Met-1 and Met-25, respectively); this might lead to leaky scanning by ribosomes to initiate translation at MDAL. In our study, our N-terminal constructs contained an optimal Kozak consensus sequence (GACCACC) before their respective initiation codons to ensure their optimal heterologous expression. Nonetheless, it is possible that the expression of the MDAL isoform was facilitated by alternative translation initiation, at the expense of expression of MANG or MSSN isoforms; therefore, additional studies are necessary to clarify the molecular mechanisms of this regulation.
      Our data suggest that the relative expression of N-terminal isoforms can act as a novel mechanism of regulation of BKCa channel activity by the auxiliary β1-subunit. Thus, our results showing the differential activity of BKCa channel N-terminal isoforms might be relevant to several tissues and pathological processes in which BKCa channels, and their β1-subunits, are involved and could lead to development of specific therapeutic strategies to regulate channel activity.

      Acknowledgments

      We thank Dr. Deborah J. Frank, Dr. Vivian Gonzalez-Perez, Prof. Christopher J. Lingle, and Prof. Jianmin Cui for critical reading of the manuscript. We also thank Prof. Jianmin Cui for kindly providing the β2-subunit construct.

      REFERENCES

        • Scott R.S.
        • Bustillo D.
        • Olivos-Oré L.A.
        • Cuchillo-Ibañez I.
        • Barahona M.V.
        • Carbone E.
        • Artalejo A.R.
        Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells.
        Pflugers Arch. 2011; 462: 545-557
        • Yazejian B.
        • Sun X.P.
        • Grinnell A.D.
        Tracking presynaptic Ca2+ dynamics during neurotransmitter release with Ca2+-activated K+ channels.
        Nat. Neurosci. 2000; 3: 566-571
        • Essin K.
        • Gollasch M.
        • Rolle S.
        • Weissgerber P.
        • Sausbier M.
        • Bohn E.
        • Autenrieth I.B.
        • Ruth P.
        • Luft F.C.
        • Nauseef W.M.
        • Kettritz R.
        BK channels in innate immune functions of neutrophils and macrophages.
        Blood. 2009; 113: 1326-1331
        • Korovkina V.P.
        • Brainard A.M.
        • England S.K.
        Translocation of an endoproteolytically cleaved maxi-K channel isoform: mechanisms to induce human myometrial cell repolarization.
        J. Physiol. 2006; 573: 329-341
        • Brayden J.E.
        • Nelson M.T.
        Regulation of arterial tone by activation of calcium-dependent potassium channels.
        Science. 1992; 256: 532-535
        • Cui J.
        • Cox D.H.
        • Aldrich R.W.
        Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels.
        J. Gen. Physiol. 1997; 109: 647-673
        • Tian L.
        • Duncan R.R.
        • Hammond M.S.
        • Coghill L.S.
        • Wen H.
        • Rusinova R.
        • Clark A.G.
        • Levitan I.B.
        • Shipston M.J.
        Alternative splicing switches potassium channel sensitivity to protein phosphorylation.
        J. Biol. Chem. 2001; 276: 7717-7720
        • Chen L.
        • Tian L.
        • MacDonald S.H.
        • McClafferty H.
        • Hammond M.S.
        • Huibant J.M.
        • Ruth P.
        • Knaus H.G.
        • Shipston M.J.
        Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) α-subunits generated from a single site of splicing.
        J. Biol. Chem. 2005; 280: 33599-33609
        • Erxleben C.
        • Everhart A.L.
        • Romeo C.
        • Florance H.
        • Bauer M.B.
        • Alcorta D.A.
        • Rossie S.
        • Shipston M.J.
        • Armstrong D.L.
        Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.
        J. Biol. Chem. 2002; 277: 27045-27052
        • Wallner M.
        • Meera P.
        • Toro L.
        Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14922-14927
        • Morrow J.P.
        • Zakharov S.I.
        • Liu G.
        • Yang L.
        • Sok A.J.
        • Marx S.O.
        Defining the BK channel domains required for β1-subunit modulation.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5096-5101
        • Tanaka Y.
        • Meera P.
        • Song M.
        • Knaus H.G.
        • Toro L.
        Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α + β subunit complexes.
        J. Physiol. 1997; 502: 545-557
        • McManus O.B.
        • Helms L.M.
        • Pallanck L.
        • Ganetzky B.
        • Swanson R.
        • Leonard R.J.
        Functional role of the β subunit of high conductance calcium-activated potassium channels.
        Neuron. 1995; 14: 645-650
        • Orio P.
        • Torres Y.
        • Rojas P.
        • Carvacho I.
        • Garcia M.L.
        • Toro L.
        • Valverde M.A.
        • Latorre R.
        Structural determinants for functional coupling between the β and α subunits in the Ca2+-activated K+ (BK) channel.
        J. Gen. Physiol. 2006; 127: 191-204
        • Wallner M.
        • Meera P.
        • Ottolia M.
        • Kaczorowski G.J.
        • Latorre R.
        • Garcia M.L.
        • Stefani E.
        • Toro L.
        Characterization of and modulation by a β-subunit of a human maxi KCa channel cloned from myometrium.
        Recept Channels. 1995; 3: 185-199
        • McCobb D.P.
        • Fowler N.L.
        • Featherstone T.
        • Lingle C.J.
        • Saito M.
        • Krause J.E.
        • Salkoff L.
        A human calcium-activated potassium channel gene expressed in vascular smooth muscle.
        Am. J. Physiol. 1995; 269: H767-H777
        • Contreras G.F.
        • Neely A.
        • Alvarez O.
        • Gonzalez C.
        • Latorre R.
        Modulation of BK channel voltage gating by different auxiliary β subunits.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 18991-18996
        • Nimigean C.M.
        • Magleby K.L.
        Functional coupling of the β(1) subunit to the large conductance Ca2+-activated K+ channel in the absence of Ca2+. Increased Ca2+ sensitivity from a Ca2+-independent mechanism.
        J. Gen. Physiol. 2000; 115: 719-736
        • Lee U.S.
        • Shi J.
        • Cui J.
        Modulation of BK channel gating by the β2 subunit involves both membrane-spanning and cytoplasmic domains of Slo1.
        J. Neurosci. 2010; 30: 16170-16179
        • Nimigean C.M.
        • Magleby K.L.
        The β subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states.
        J. Gen. Physiol. 1999; 113: 425-440
        • Knaus H.G.
        • Garcia-Calvo M.
        • Kaczorowski G.J.
        • Garcia M.L.
        Subunit composition of the high conductance calcium-activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels.
        J. Biol. Chem. 1994; 269: 3921-3924
        • Wang Y.W.
        • Ding J.P.
        • Xia X.M.
        • Lingle C.J.
        Consequences of the stoichiometry of Slo1 α and auxiliary β subunits on functional properties of large-conductance Ca2+-activated K+ channels.
        J. Neurosci. 2002; 22: 1550-1561
        • Wallner M.
        • Meera P.
        • Toro L.
        Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane β-subunit homolog.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4137-4142
        • Orio P.
        • Latorre R.
        Differential effects of β1 and β2 subunits on BK channel activity.
        J. Gen. Physiol. 2005; 125: 395-411
        • Yang H.
        • Zhang G.
        • Shi J.
        • Lee U.S.
        • Delaloye K.
        • Cui J.
        Subunit-specific effect of the voltage sensor domain on Ca2+ sensitivity of BK channels.
        Biophys. J. 2008; 94: 4678-4687
        • Yan J.
        • Aldrich R.W.
        BK potassium channel modulation by leucine-rich repeat-containing proteins.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7917-7922
        • Behrens R.
        • Nolting A.
        • Reimann F.
        • Schwarz M.
        • Waldschütz R.
        • Pongs O.
        hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel β subunit family.
        FEBS Lett. 2000; 474: 99-106
        • Knaus H.G.
        • Folander K.
        • Garcia-Calvo M.
        • Garcia M.L.
        • Kaczorowski G.J.
        • Smith M.
        • Swanson R.
        Primary sequence and immunological characterization of β-subunit of high conductance Ca2+-activated K+ channel from smooth muscle.
        J. Biol. Chem. 1994; 269: 17274-17278
        • Brenner R.
        • Peréz G.J.
        • Bonev A.D.
        • Eckman D.M.
        • Kosek J.C.
        • Wiler S.W.
        • Patterson A.J.
        • Nelson M.T.
        • Aldrich R.W.
        Vasoregulation by the β1 subunit of the calcium-activated potassium channel.
        Nature. 2000; 407: 870-876
        • Plüger S.
        • Faulhaber J.
        • Fürstenau M.
        • Löhn M.
        • Waldschütz R.
        • Gollasch M.
        • Haller H.
        • Luft F.C.
        • Ehmke H.
        • Pongs O.
        Mice with disrupted BK channel beta1 subunit gene feature abnormal Ca2+ spark/STOC coupling and elevated blood pressure.
        Circ. Res. 2000; 87: E53-E60
        • Petkov G.V.
        • Bonev A.D.
        • Heppner T.J.
        • Brenner R.
        • Aldrich R.W.
        • Nelson M.T.
        β1-subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle.
        J. Physiol. 2001; 537: 443-452
        • Matharoo-Ball B.
        • Ashford M.L.
        • Arulkumaran S.
        • Khan R.N.
        Down-regulation of the α- and β-subunits of the calcium-activated potassium channel in human myometrium with parturition.
        Biol. Reprod. 2003; 68: 2135-2141
        • Brainard A.M.
        • Korovkina V.P.
        • England S.K.
        Potassium channels and uterine function.
        Semin. Cell Dev. Biol. 2007; 18: 332-339
        • Chan Y.W.
        • van den Berg H.A.
        • Moore J.D.
        • Quenby S.
        • Blanks A.M.
        Assessment of myometrial transcriptome changes associated with spontaneous human labour by high throughput RNA-seq.
        Exp. Physiol. 2014; 99: 510-524
        • Cox D.H.
        • Aldrich R.W.
        Role of the β1 subunit in large-conductance Ca2+-activated K+ channel gating energetics. Mechanisms of enhanced Ca2+ sensitivity.
        J. Gen. Physiol. 2000; 116: 411-432
        • Bao L.
        • Cox D.H.
        Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its β1 subunit.
        J. Gen. Physiol. 2005; 126: 393-412
        • Sun X.
        • Shi J.
        • Delaloye K.
        • Yang X.
        • Yang H.
        • Zhang G.
        • Cui J.
        The interface between membrane-spanning and cytosolic domains in Ca2+-dependent K+ channels is involved in β subunit modulation of gating.
        J. Neurosci. 2013; 33: 11253-11261
        • Fernandez F.R.
        • Morales E.
        • Rashid A.J.
        • Dunn R.J.
        • Turner R.W.
        Inactivation of Kv3.3 potassium channels in heterologous expression systems.
        J. Biol. Chem. 2003; 278: 40890-40898
        • Simkin D.
        • Cavanaugh E.J.
        • Kim D.
        Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation.
        J. Physiol. 2008; 586: 5651-5663
        • Thomas D.
        • Plant L.D.
        • Wilkens C.M.
        • McCrossan Z.A.
        • Goldstein S.A.
        Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium.
        Neuron. 2008; 58: 859-870
        • Cai J.
        • Huang Y.
        • Li F.
        • Li Y.
        Alteration of protein subcellular location and domain formation by alternative translational initiation.
        Proteins. 2006; 62: 793-799
        • Kozak M.
        Structural features in eukaryotic mRNAs that modulate the initiation of translation.
        J. Biol. Chem. 1991; 266: 19867-19870
        • Kozak M.
        Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6.
        EMBO J. 1997; 16: 2482-2492