Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca2+ (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 2003
The 1 subunit of BK (large conductance Ca 2؉ and voltage-activated K ؉) channels is essential for many key physiological processes, such as controlling the contraction of smooth muscle and the tuning of hair cells in the cochlea. Although it is known that the 1 subunit greatly increases the open probability of BK channels, little is known about its mechanism of action. We now explore this mechanism by using channels in which the Ca 2؉-and Mg 2؉dependent activating mechanisms have been disrupted by mutating three sites to remove the Ca 2؉ and Mg 2؉ sensitivity. We find that the presence of the 1 subunit partially restores Ca 2؉ sensitivity to the triply mutated channels, but not the Mg 2؉ sensitivity. We also find that the 1 subunit has no effect on the Mg 2؉ sensitivity of WT BK channels, in contrast to its pronounced effect of increasing the apparent Ca 2؉ sensitivity. These observations suggest that the 1 subunit increases open probability by working through the Ca 2؉-dependent, rather than Mg 2؉-dependent, activating mechanisms, and that the action of the 1 subunit is not directly on the Ca 2؉ binding sites, but on the allosteric machinery coupling the sites to the gate. The differential effects of the 1 subunit on the Ca 2؉ and Mg 2؉ activation of the channel suggest that these processes act separately. Finally, we show that Mg i 2؉ inhibits, rather than activates, BK channels in the presence of the 1 subunit for intermediate levels of Ca i 2؉. This Mg 2؉ inhibition in the presence of the 1 subunit provides an additional regulatory mechanism of BK channel activity.
The Journal of General Physiology, 2000
Coexpression of the β1subunit with the α subunit (mSlo) of BK channels increases the apparent Ca2+sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca2+sensitivity requires Ca2+, by comparing the gating in 0 Ca2+iof BK channels composed of α subunits to those composed of α+β1subunits. The β1subunit increased burst duration ∼20-fold and the duration of gaps between bursts ∼3-fold, giving an ∼10-fold increase in open probability (Po) in 0 Ca2+i. The effect of the β1subunit on increasing burst duration was little changed over a wide range ofPoachieved by varying either Ca2+ior depolarization. The effect of the β1subunit on increasing the durations of the gaps between bursts in 0 Ca2+iwas preserved over a range of voltage, but was switched off as Ca2+iwas increased into the activation range. The Ca2+-independent, β1subunit-induced increase in burst duration accounted for 80% of the leftward shift in thePovs. Ca2+icurve that reflects the i...
The Journal of General Physiology, 1999
Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+(BK) channels greatly increases the apparent Ca2+sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+sensitivity of the channel. In the presence of the β subunit, each burst of ope...
Mechanism of 4 Subunit Modulation of BK Channels
The Journal of General Physiology, 2006
Large-conductance (BK-type) Ca 2+ -activated potassium channels are activated by membrane depolarization and cytoplasmic Ca 2+ . BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specifi c accessory subunits (β1-β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca 2+ sensitivity. Specifi cally, channel activity at low Ca 2+ is inhibited, while at high Ca 2+ , activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in P o (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of P o -V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we fi nd these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L 0 ), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh o ) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vh o and an increase in allosteric coupling to Ca 2+ binding (C) promotes a negative G-V shift of α+β4 channels as compared to α subunits alone. This manner of modulation predicts that type II BK channels are downregulated by β4 at resting voltages through effects on L 0 . However, β4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.
A BK (Slo1) channel journey from molecule to physiology
Channels, 2013
Abbreviations: BK, big conductance voltage and Ca 2+ -dependent potassium channel; Charybdotoxin, ChTx; Iberotoxin, IbTx; regulator of the conductance of K + channels, RCK; voltage sensing domain, VSD; leucine-rich repeat proteins, LRRC; nitric oxide, NO; cyclic guanosin mono-phosphate, cGMP www.landesbioscience.com Channels 443
Elimination of the BK Ca Channel's High-Affinity Ca 2 � Sensitivity
We report here a combination of site-directed mutations that eliminate the high-affinity Ca 2 ϩ response of the large-conductance Ca 2 ϩ -activated K ϩ channel (BK Ca ), leaving only a low-affinity response blocked by high concentrations of Mg 2 ϩ . Mutations at two sites are required, the "Ca 2 ϩ bowl," which has been implicated previously in Ca 2 ϩ binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK Ca channel contains three types of Ca 2 ϩ binding sites, one of low affinity that is Mg 2 ϩ sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca 2 ϩ to influence channel opening. Estimates of the binding characteristics of the BK Ca channel's high-affinity Ca 2 ϩ -binding sites are provided.
Differential Effects of 1 and 2 Subunits on BK Channel Activity
The Journal of General Physiology, 2005
High conductance, calcium-and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (  ) subunits.  1 and  2 subunits increase apparent channel calcium sensitivity. The  1 subunit also decreases the voltage sensitivity of the channel and the  2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the  1 and  2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage . In this study, we used a  2 subunit without its N-type inactivation domain (  2IR). The results indicate that the  2IR subunit, like the  1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the  1 subunit, the  2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied  subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the  1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both  subunits reduce the intrinsic equilibrium constant for channel opening ( L 0 ). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the  1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the ␣ subunit is coexpressed with the  2IR subunit.
Calcium-driven regulation of voltage-sensing domains in BK channels
2019
Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore domain govern the Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the Ca2+- and voltage-sensing mechanisms crosstalk on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation measuring and analyzing the effects of internal Ca2+ on BK channels gating currents. Our results indicate that the Ca2+ sensors occupancy has a strong impact on the VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, appear to contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between C...