Ca ²⁺ -binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca ²⁺ -dependent activation

Abstract

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca 2؉ -activated K ؉ channel (BK channel) contains Ca 2؉ -binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca 2؉ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG-and His 6-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a 45 Ca 2؉ -overlay protein blot assay to detect Ca 2؉ binding. Dslo-C280 exhibits specific binding of 45 Ca 2؉ in comparison with various control proteins and known EF-hand Ca 2؉ -binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the ''Ca-bowl'' motif are changed to Asn, reduces 45 Ca 2؉ -binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca 2؉ sensitivity for activation and a shift of Ϸ؉80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca 2؉ and a reduction in apparent Ca 2؉ affinity, suggesting the loss of one Ca 2؉ -binding site per monomer. These results demonstrate a functional correlation between Ca 2؉ binding to a specific region of the BK protein and Ca 2؉ -dependent activation, thus providing a biochemical approach to study this process.

The RCK1 high-affinity Ca 2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels

“…At least two high-affinity Ca 2ϩ sensors in each Slo1 polypeptide are known: the RCK1 sensor encompassing D367 (27) and the Ca 2ϩ bowl sensor in the RCK2 domain (28,29). Inspection of the human Slo1 amino acid sequence showed that the putative RCK1 Ca 2ϩ sensor subdomain contains several histidine residues near D367, a critical component of the RCK1 high-affinity Ca 2ϩ sensor (27) (Fig.…”

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca 2+

“…In this guardian role of controlling Ca 2ϩ influx and excitability, BK channels modulate many processes, such as spike broadening in neurons (3), smooth muscle contraction (4), neurotransmitter release (5), and the electrical tuning of hair cells (6,7). The activation of BK channels by Ca i 2ϩ typically occurs with Hill coefficients of 2-5 (1,(8)(9)(10)(11), indicating that Ca i 2ϩ acts in a highly cooperative manner. These coefficients suggest that a minimum of 2-5 Ca 2ϩ must be bound to allosteric activators to maximally activate the channel.…”

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

“…1A, C). Partial deletion or point mutations of the aspartates contained in the calcium bowl produced BK channel that were less sensitive to Ca 2+ -at the same Ca 2+ concentration, the calcium bowl mutant conductance-voltage (G-V) curves were right-shifted compared to the wildtype BK channel G-V curve (Schreiber and Salkoff, 1997;Bian et al, 2001;Braun and Sy, 2001). If the Ca 2+ bowl is disrupted by deleting crucial aspartates, this high Ca 2+ affinity regulatory site is lost, but the mutant and the wild-type BK showed the same sensitivity to Cd 2+ ions.…”

“…The D5N5 mutant expressed in cells exhibits a lower Ca 2+ sensitivity for activation and reduces the dSlo channel's Hill coefficient 2-fold, as if one binding site per monomer is lost in the D5N5 mutant. The most economic way to account for these results is to assume the existence of two distinct Ca 2+ -binding sites per channel monomer (Bian et al, 2001;cf. Schreiber and Salkoff, 1997).…”

“…A third, low affinity mechanism (discussed below), can be put into action by Ca 2+ , Sr 2+ , Cd 2+ , Mn 2+ , Ni 2+ , and Co 2+ . In the absence of binding assays like those performed with BK carboxyl terminus fusion proteins (Bian et al, 2001;Braun and Sy, 2001), there exists the possibility, however, that all the results are due to coincidental allosteric effects on Ca 2+ binding to a region that lies outside the RCK region. Independent of these considerations, the unique divalent cation selectivity of the different BK regulatory mechanisms supports the existence of at least three distinct divalent cation binding sites and that the sites act independently of each other.…”