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

Abstract

We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca 2؉ . Moreover, in twohybrid assays where it is possible to detect interactions with apo-CaM but not with Ca 2؉ -bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two ␣-helices (A and B). These two helices encompass ϳ85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of ϳ130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Fulllength CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca 2؉ levels and that this interaction is disturbed when the [Ca 2؉ ] is raised. Thus, we propose that CaM acts as a mediator in the Ca 2؉ -dependent modulation of KCNQ channels.The KCNQ transmembrane proteins are members of a family of voltage-dependent potassium selective channels that are involved in the control of cellular excitability. Remarkably, mutations in four of the five known members of this family have been associated with different hereditary human disorders. While mutations in the KCNQ1 subunit (KvQT1) lead to arrhythmia in the human long QT syndrome, mutations in KCNQ2 or KCNQ3 are associated with a benign form of epilepsy. It has also been shown that KCNQ4 is mutated in a dominant form of progressive hearing loss (1).With regards to the normal physiology of this protein family, the KCNQ2 and KCNQ3 subunits have been shown to form M-type potassium channels whose expression is restricted to neuronal tissue (2). Moreover, in some brain areas and neuronal tissues, KCNQ4 and KCNQ5 also contribute to the formation of M channels, suggesting that the different combinations of KCNQ subunits may be in part responsible for the diversity of M channel properties (1). The M current (I M ) is a subthreshold noninactivating voltage-dependent potassium current that is found in many neuronal cell types. The M current controls membrane excitability, and it has been shown to be modulated by a variety of intracellular signals that in turn dramatically affect the firing rate of neurons. Among those intracellular signals, Ca 2ϩ has been shown to mediate the inhibition of I M by B 2 bradykinin receptors in sympathetic neurons (3). Indeed, intracellular Ca 2ϩ can suppress the activity of M channels under conditions that do not support enzymatic activities such as phosphorylation (4). Th...

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

“…CaM binds the non-continuous domain formed by helix A and B of Kv7 channels [6,26]; (Figure 1(a)). Since both neutralizing mutations are adjacent to the interaction site (Figure 1(a,b)), we tested how CaM binding was affected.…”

“…GST-Kv7.2 Helices A-B fusion protein carrying the K526N mutation was purified. Other mutations, (A343D, I340E in helix A and S511D in helix B), known to disrupt CaM binding [6], or to cause BFNE (R353G) [15,35], were used as negative controls, (Figure 4(a)). We also examined two CaM binding proteins fused to GST as positive controls for the interaction in the presence (the C-terminus of the NMDA receptor, NR1a) or absence (neurogranin) of Ca 2+ [6,18].…”

“…Other mutations, (A343D, I340E in helix A and S511D in helix B), known to disrupt CaM binding [6], or to cause BFNE (R353G) [15,35], were used as negative controls, (Figure 4(a)). We also examined two CaM binding proteins fused to GST as positive controls for the interaction in the presence (the C-terminus of the NMDA receptor, NR1a) or absence (neurogranin) of Ca 2+ [6,18]. Fusion proteins were immobilized on GSH-Sepharose beads and incubated with CaM, both in the presence and absence of Ca 2+ , and anti-CaM antibodies were then used in Western blot to detect the AB-CaM interaction (see “Materials and methods” section).…”

“…(a), Cartoon representation of a Kv7.2 subunit. The amino acids corresponding to helices A and B are shadowed in green [6,26]. The CaM binding domains are indicated in blue, while putative residues of Helix A [28,29] and Helix B [30] implicated in the interaction with PIP 2 are boxed in brown.…”

“…CaM, whose binding site is created by helices A and B in the C-terminus (Figure 1(a)), is required to produce functional Kv7.2 channels [5,6]. CaM affects not only PIP 2 sensitivity [7,8], but also influences voltage sensitivity [7,9,10], regulation of the stability of the distal tetramerization domain [11], functional connection between this distal coiled-coil tetramerization domain and PIP 2 modulation [12] and plays a role in the suppression of Kv7.2/Kv7.3 currents mediated by bradykinin [13,14].…”