The NH2 terminus of RCK1 domain regulates Ca2+-dependent BKCa channel gating

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, United States
The Journal of General Physiology (Impact Factor: 4.79). 10/2005; 126(3):227-41. DOI: 10.1085/jgp.200509321
Source: PubMed


Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.

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Available from: Gayathri Krishnamoorthy
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    • "Taken these results together, it is likely that the C-Linker and AC region of Slo1 may interact with the cytosolic domain of the β2 subunit either directly or through an allosteric mechanism. Previous studies show that changes in the length of the C-Linker alter Ca 2+ sensitivity of the channel (Niu et al., 2004), while mutations in the AC region revealed an allosteric network important for Ca 2+ dependence of channel gating (Krishnamoorthy et al., 2005;Yang et al., 2010). Our results further support the important role of the C-Linker and AC region in mediating the coupling between Ca 2+ binding and channel opening and show that the β2 subunit may modulate Ca 2+ sensitivity by altering such coupling. "
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    ABSTRACT: Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory β subunits. Four types of BK channel β subunits (β1-β4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the β1 and β2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these β subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for β2 modulation based on the study of differential effects of the β2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (βA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the β2 but not for the β1 modulation. These results suggest that the β2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel β subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.
    Full-text · Article · Dec 2010 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    • "), and is important in determining Ca 2+ sensitivity (Krishnamoorthy et al., 2005). Furthermore, both the putative Ca 2+ -binding site D367 and the mutation mD369G are situated within the AC region so that the mutation may alter its structure to affect Ca 2+ sensitivity. "
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    ABSTRACT: Ca(2+)-activated BK channels modulate neuronal activities, including spike frequency adaptation and synaptic transmission. Previous studies found that Ca(2+)-binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca(2+) binding opens the gate over this distance remains unknown. By studying an Asp-to-Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca(2+) binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca(2+) sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca(2+) binding to channel opening.
    Full-text · Article · Jun 2010 · Neuron
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    • "Moreover, Slo1 channels can coimmunoprecipitate with actin (Brainard et al., 2005), and the cytosolic domains in Slo1 can bind to cortactin, an actin-binding scaffolding protein (Tian et al., 2006). This latter interaction seems to be essential for stretch-induced activation of BK Ca channels, and it occurs in a cytosolic region of the channel close to a domain known to play a role in Ca 2 -dependent gating (Krishnamoorthy et al., 2005; Zeng et al., 2005; Tian et al., 2006). Much less is known about long-term regulation of BK Ca channels. "
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    ABSTRACT: Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the physiological properties of many cell types. The gating properties of BK(Ca) channels are Ca(2+)-, voltage- and stretch-sensitive, and stretch-sensitive gating of these channels requires interactions with actin microfilaments subjacent to the plasma membrane. Moreover, we have previously shown that trafficking of BK(Ca) channels to the plasma membrane is associated with processes that alter cytoskeletal dynamics. Here, we show that the Slo1 subunits of BK(Ca) channels contain a novel cytoplasmic actin-binding domain (ABD) close to the C terminus, considerably downstream from regions of the channel molecule that play a major role in determining channel-gating properties. Binding of actin to the ABD can occur in a binary mixture in the absence of other proteins. Coexpression of a small ABD-green fluorescent protein fusion protein that competes with full-length Slo1 channels for binding to actin markedly suppresses trafficking of full-length Slo1 channels to the plasma membrane. In addition, Slo1 channels containing deletions of the ABD that eliminate actin binding are retained in intracellular pools, and they are not expressed on the cell surface. At least one point mutation within the ABD (L1020A) reduces surface expression of Slo1 channels to approximately 25% of wild type, but it does not cause a marked effect on the gating of point mutant channels that reach the cell surface. These data suggest that Slo1-actin interactions are necessary for normal trafficking of BK(Ca) channels to the plasma membrane and that the mechanisms of this interaction may be different from those that underlie F-actin and stretch-sensitive gating.
    Preview · Article · Mar 2008 · Molecular pharmacology
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