Hydrophobic Interface between Two Regulators of K+ Conductance Domains Critical for Calcium-dependent Activation of Large Conductance Ca2+-activated K+ Channels

Abstract

It has been suggested that the large conductance Ca 2؉ -activated K ؉ channel contains one or more domains known as regulators of K ؉ conductance (RCK) in its cytosolic C terminus. Here, we show that the second RCK domain (RCK2) is functionally important and that it forms a heterodimer with RCK1 via a hydrophobic interface. Mutant channels lacking RCK2 are nonfunctional despite their tetramerization and surface expression. The hydrophobic residues that are expected to form an interface between RCK1 and RCK2, based on the crystal structure of the bacterial MthK channel, are well conserved, and the interactions of these residues were confirmed by mutant cycle analysis. The hydrophobic interaction appears to be critical for the Ca 2؉ -dependent gating of the large conductance Ca 2؉ -activated K ؉ channel.Large conductance calcium-activated potassium (BK Ca ) 2 channels play a key role in modulating a number of important physiological processes, such as neuronal excitability, frequency tuning of hair cells, smooth muscle contraction, and immunity (1-9). BK Ca channels are activated by membrane depolarization and an increase in intracellular calcium (10 -13). Thus, BK Ca channels are considered to be molecular integrators of biochemical and electrical signals. Membrane depolarization and calcium binding activate BK Ca channels independently via separate regions of the ␣ subunit of the channel (Slo). The transmembrane segments of the Slo channel, S1-S6, are structurally similar to those of voltage-gated potassium channels, and as in these channels, charged residues in the Slo S1-S4 segments are thought to be involved in the voltage-dependent gating of the channel (14 -20). It is generally accepted that the bulkycytoplasmicCterminusofSloisresponsibleforthecalciumdependent activation of the channel (21-23).The cytoplasmic C terminus of Slo has been proposed to contain more than two Ca 2ϩ -sensing sites, a high affinity site called the Ca 2ϩ bowl, a low affinity site, and additional high affinity sites within a structural module known as the regulator of K ϩ conductance (RCK) domain (22, 24 -29). The Ca 2ϩ bowl is composed of a series of Asp residues and binds Ca 2ϩ with micromolar affinity. Mutations here have been shown to cause positive shifts in the conductance-voltage (G-V) relationship at constant [Ca 2ϩ ], which are similar to those observed with the wild-type channel when [Ca 2ϩ ] is lowered (22,23,26,30,31). The RCK domain is found primarily in prokaryotic ligandgated K ϩ channels and in some bacterial K ϩ uptake and efflux systems, in which it is also called the K ϩ transport nucleotidebinding (KTN) domain (25,(32)(33)(34). Crystal structures of RCK domains have been determined from Ca 2ϩ -activated K ϩ channels in Escherichia coli and Methanobacterium thermoautotrophicum (24,25,35,36). The structure of the tetrameric MthK channel shows that an octameric complex could be formed by intermolecular interactions on fixed and flexible interfaces between a tetramer of dimeric RCK domains. This complex, called the...

Profiling the Phospho-status of the BKCa Channel α Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

“…Membrane topology of BK␣ together with localization of the identified phosphosites and splice insertions. The constitutive form of BK␣ contains the transmembrane core (S0 -S6), the pore region, the hydrophobic intracellular segments S7-S10, the Ca 2ϩ bowl, and the RCK domains (25,65,66). Insertion sites of sequence stretches generated by alternative splicing or alternative start of translation are indicated by triangles with the same color coding and names as in Fig.…”

“…Five phosphorylation sites (Thr(P)-640, Ser(P)-642, Ser(P)-655, Thr(P)-658, and Ser(P)-659) were clustered between hydrophobic segments S8 and S9 immediately following the Strex splice site. This region has been suggested to form a flexible linker between the two RCK domains (25,47). Seven phosphorylation sites were found in the putative RCK2 domain, with Ser(P)-777 located near the C-terminal end of S9, and Ser(P)-843, Thr(P)-847, Ser(P)-854, Ser(P)-855, Ser(P)859, and Ser(P)-869 clustered near the Ca 2ϩ -bowl and the beginning of S10.…”

“…1). This C-terminal domain comprises Ͼ70% of the total protein and contains four hydrophobic segments (S7-S10) and sequences similar to RCK (Regulating Conductance of K ϩ ) domains (23)(24)(25), and a string of Asp residues known as the "Ca 2ϩ bowl" (26). BK␣ contains ϳ200 serine and threonine residues that can be potentially phosphorylated by cellular protein kinases (supplemental material).…”

α5β1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

“…In this regard, Tyr-766 is predicted to reside within the putative RCK2 domain of the channel, which appears to interact functionally with the more proximal RCK1 domain to affect channel gating (46). The observation that V 0.5 did not shift in 0 Ca 2ϩ after integrin activation or with c-src overexpression is consistent with this idea and with the previous work of Ling et al (7) who showed that c-src activity did not affect channel gating properties at very low (Ͻ0.9 M) Ca 2ϩ levels.…”

Mg 2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels

“…Physiological concentrations of Mg 2ϩ in the low millimolar range activate BK channels, independent of the effects of micromolar Ca 2ϩ , by binding to a site that includes residues E374 and E399 (19-21). The putative Mg 2ϩ binding site is located in a cytosolic domain that is homologous to the RCK1 domain of MthK and Escherichia coli K ϩ channels (19,(22)(23)(24)(25). Fig.…”