BK_Ca α-subunit with distinct N-termini are differentially modulated by β1-β2 chimeric constructs.

<p>Representative inside-out single-channel recordings from HEK293T cells transfected with MANG (<b>A</b>), MSSN (<b>D</b>) or MDAL (<b>G</b>) in the presence or absence of β1β2β1 or β2NDβ1β2, at different membrane potentials (-40 mV to +40 mV) with 10 μM Ca²⁺ in the bath. Dashed lines indicate closed (C) states of the channels. Voltage-activation of MANG (<b>B</b> and <b>C</b>), MSSN (<b>E</b> and <b>F</b>) or MDAL (<b>H</b> and <b>I</b>), in the absence (α, black symbols) or presence of β1 (blue symbols), β2ND (red symbols), β1β2β1 (orange symbols) or β2NDβ1β2 (purple symbols), expressed as open probability (<i>P</i>_<i>o</i>) of the channel, in the presence of 10 μM (<b>B</b>, <b>E</b> and <b>H</b>) or 100 μM (<b>C</b>, <b>F</b>, and <b>I</b>) Ca²⁺ in the bath; n = 3–19, symbols are mean ± SEM.</p

Effect of β2ND,β1β2β1 and β2NDβ1β2 constructs on the open dwell-times of BK_Ca channels with distinctive N-terminal regions.

<p>Effect of β2ND,β1β2β1 and β2NDβ1β2 constructs on the open dwell-times of BK_Ca channels with distinctive N-terminal regions.</p

Effects of β2ND and β1-β2 chimeric constructs on BK_Ca α-subunit N-terminal constructs single-channel kinetics.

<p>Open (<b>A</b>) and (<b>B</b>) closed dwell-times distribution histograms of single-channels in HEK293T cells expressing MANG, MSSN or MDAL constructs. Patches containing channels composed by α-subunit alone (black lines), α+β2ND (red lines) α+β1β2β1 (orange lines) or α+β2β1β2 (purple lines) were analyzed (n = 6–11). Single-channel currents were elicited by holding the membrane potential at a certain voltage (-20 mV to -100 mV) for at least 1 min in the presence of 10 μM Ca²⁺ in the bath. Histograms were plotted in log-bin timescales and fitted with double exponential functions.</p

Modulation of voltage-dependent activation in BK_Ca α-subunit with distinct N-termini by β-subunit constructs.

<p>Analysis of the voltage of half maximal activation (<i>V</i>_{<i>0</i>.<i>5</i>}) of the N-terminal BK_Ca constructs MANG (<b>A</b>), MSSN (<b>B</b>) or MDAL (<b>C</b>), in the absence (α, black symbols) or presence of β1 (blue symbols), β2ND (red symbols), β1β2β1 (orange symbols) or β2NDβ1β2 (purple symbols), at different [Ca²⁺] in the bath. Symbols are mean ± SEM.</p

The unique N-terminal sequence of the BK_Ca channel α-subunit determines its modulation by β-subunits

<div><p>Large conductance voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BK_Ca channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BK_Ca channels is the β1-subunit. We have previously described that BK_Ca channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the β1-subunit, but not by the β2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by β-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BK_Ca containing three N-terminal sequences with two β1-β2 chimeric constructs containing the extracellular loop of β1 or β2, and the transmembrane and cytoplasmic domains of β2 or β1, respectively. Both β chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 μM intracellular Ca²⁺. However, MSSN showed no shift of the voltage-activation, at the same Ca²⁺ concentrations. The presence of the extracellular loop of β1 in the chimera resembled results seen with the full β1 subunit, suggesting that the extracellular region of β1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BK_Ca channels either containing or lacking this poly-serine stretch were leftward shifted by β1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BK_Ca channel by β-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.</p></div