Domains Responsible for Constitutive and Ca²⁺-Dependent Interactions between Calmodulin and Small Conductance Ca²⁺-Activated Potassium Channels

Abstract

Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca(2+)-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca(2+)-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca(2+)-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca(2+)-dependent and Ca(2+)-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

The Identification and Characterization of a Noncontinuous Calmodulin-binding Site in Noninactivating Voltage-dependent KCNQ Potassium Channels

“…The binding of Ca 2ϩ to the EF hands results in the creation of a surface that serves as an interface for the association of CaM with the target protein. Each lobe can control distinct processes in the target protein (21,22), and binding to a target can change the affinity for Ca 2ϩ (23).…”

“…These Asp to Ala mutations in the EF hands greatly diminish or abolish their ability to bind Ca 2ϩ (21,24,25). Both mutants were able to interact with the C-terminal region of KCNQ2 or KCNQ3, although less strongly than wild type CaM.…”

Regional Differences in Distribution and Functional Expression of Small-Conductance Ca2+-Activated K+Channels in Rat Brain

“…When expressed in Xenopus oocytes, the resulting channels are voltage-insensitive and activated by submicromolar intracellular Ca 2ϩ (Kohler et al, 1996;Hirschberg et al, 1998). Structurefunction analysis revealed that their Ca 2ϩ gating is mediated by constitutive association with calmodulin (Xia et al, 1998;Keen et al, 1999). However, direct interaction of calmodulin with native brain SK channels so far has been demonstrated only for SK3 (Xia et al, 1998).…”

Orai1 pore residues control CRAC channel inactivation independently of calmodulin

“…Therefore, we tested CaM 12 and CaM 34 mutant proteins, in which Ca 2+ binding to only the N-or C-lobe EF hands is altered, respectively. Because the CaM 12 mutation is known to suppress rSK2 current in a manner similar to CaM 1234 (Keen et al, 1999), we used an experimental design identical to that described in the preceding paragraph for CaM 1234 . Like CaM 1234 , CaM 12 effectively suppressed rSK2 current ( Fig.…”

“…The CaM 34 mutant does not affect rSK2 gating (Keen et al, 1999), but it is known to suppress CDI of the voltage-gated channel Ca V 1.2 (Peterson et al, 1999). Thus, as a positive control for knockdown of CaM function by CaM 34 , we compared inactivation of Ca V 1.2 currents carried by Ca 2+ and Ba 2+ (which supports only voltagedependent inactivation; see Materials and methods and Fig.…”