Locations of the β1 transmembrane helices in the BK potassium channel

Abstract

BK channels are composed of ␣-subunits, which form a voltageand Ca 2؉ -gated potassium channel, and of modulatory ␤-subunits. The ␤1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca 2؉ ]i in a voltage range near the smoothmuscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltagedependent Ca 2؉ channels and of smooth muscle tone. We explored the contacts between ␣ and ␤1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two ␤1 transmembrane (TM) helices, TM1 and TM2, and to the seven ␣ TM helices, consisting of S1-S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1-S4. We now find that the extracellular ends of ␤1 TM2 and ␣ S0 are in contact and that ␤1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3-S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance-voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N-and C-terminal tails of ␤1, to the modulation of BK channel function.auxiliary subunit ͉ cysteine substitution ͉ disulfide cross-linking ͉ electrophysiology ͉ mslo1 B K potassium channels have large conductances and are uniquely both voltage-and Ca 2ϩ -activated. They are negative feedback regulators of cellular excitability and of [Ca 2ϩ ] i . BK channels are a complex of four ␣-subunits (1) and four ␤-subunits (2-7). In addition to the S1-S6 transmembrane (TM) helices conserved in all voltage-gated K ϩ channels, ␣ contains a unique seventh TM helix, S0, N-terminal to S1-S6; furthermore, the first 19 residues preceding S0 are extracellular (Fig. 1A) (8).A large cytoplasmic domain, C-terminal to S6, contains two RCK domains, resembling those of the bacterial Ca 2ϩ -gated K ϩ channel, MthK (9). The voltage-sensitivity of the BK channel is a property of the membrane domain of ␣, whereas the Ca 2ϩ -sensitivity is conferred by the C-terminal cytoplasmic domain (10).The channel formed by BK ␣ is modulated by ␤-subunits. There are four types of ␤-subunits, ␤1, ␤2, ␤3, and ␤4, 191-235 residues in length (2-6). ␤-Subunits have short cytoplasmic Nand C-terminal segments, two TM helices, TM1 and TM2, and a large extracellular loop between them (Fig. 1B). The ␤1-subunit is expressed in smooth muscle. In the presence of Ca 2ϩ , it shifts the V 50 for channel activation toward the resting potential, around which channel opening becomes a roughly linear function of [Ca 2ϩ ] i in the physiologically relevant 1-10 M range. In addition, ␤1 slows both activation and deactivation. Activation of BK channels shifts the membrane potential to more negative values, suppressing the activity of L-ty...

Location of KCNE1 relative to KCNQ1 in the I KS potassium channel by disulfide cross-linking of substituted cysteines

“…Disulfide crosslinking has been widely used to determine quaternary structures of proteins and to determine regions that move during changes in functional states (24). Using our variation of this approach (25,26), we substituted Cys, one at a time, in the first four positions just flanking the extracellular ends of Q1 S1-S6 and just flanking the extracellular end of the E1-TM. We expressed 80 distinct pairs of Cys-substituted Q1 and Cys-substituted E1 in CHO cells and determined the extents of spontaneous disulfide bond formation between Q1 and E1 in channels expressed on the cell surface.…”

The BK channel: a vital link between cellular calcium and electrical signaling

“…Direct structural interaction between the β1 subunit and the BK voltage-sensing domain is supported by functional and biochemical analysis, which has demonstrated a key role for the S0 transmembrane helix and suggested that there are direct molecular contacts between S0 and the second transmembrane segment (TM2) of the β1 subunit, and between S1 and S2 and the first transmembrane segment (TM1) (Wallner et al, 1996;Morrow et al, 2006;Liu et al, 2008). Similarly, gating effects of the neuronal β4 subunit could be explained by stabilization of the BK channel voltage sensor in the activated state, in combination with relative stabilization of the pore in the closed state in the absence of voltage-sensor activation, giving rise to a gating phenotype in which the voltage-dependent activation is shift toward more positive voltages at low [Ca 2+ ] and more negative voltages at higher [Ca 2+ ] in channels comprised of alpha + β4 subunits (Wang et al, 2006;Wu et al, 2009).…”

“…The S1-S6 segments are analogous to the S1-S6 of the other Kv channels; the S5-S6 segments line the pore, and the S1-S4 segments contribute to "sensing" the transmembrane voltage, by way of a series of charged residues in the S4 region. The additional and relatively unique S0 region gives the BK channel an extracellular N-terminus, and forms a functionally important interaction with the β1 subunit (Wallner et al, 1996;Meera et al, 1997;Morrow et al, 2006;Liu et al, 2008). The S0 segment also forms an integral part of the BK channel voltage-sensor domain (Koval et al, 2007;Pantazis et al, 2010).…”

“…the BK voltage-sensing domain (Morrow et al, 2006;Liu et al, 2008;Wu et al, 2009). Both β and γ subunits exhibit tissue-specific expression, and impose an array functional effects on BK channel activation properties, which can be inhibitory (β2 and β3), facilitatory (β1, γ1-4), or mixed (β4) (summarized in Table 1) (Nimigean and Magleby, 1999;Brenner et al, 2000a;Cox and Aldrich, 2000;Nimigean and Magleby, 2000;Bao and Cox, 2005;Wang et al, 2006;Yan and Aldrich, 2012).…”

Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

“…The auxiliary β1 subunit of BK channels alters the voltage-dependence by interacting with S0, S1 and S2 in the periphery of the VSD [210,211].…”