Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

Abstract

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K ؉ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca 2؉ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca 2؉ sensor, conferring Ca 2؉ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

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

“…Some mutations linked to BFNE cause ER retention and a consequent reduction in surface expression due to disruption of CaM binding [15,18]. CaM binds the non-continuous domain formed by helix A and B of Kv7 channels [6,26]; (Figure 1(a)).…”

“…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).…”

“…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). Pull-down assays showed that in the presence of Ca 2+ (Figure 4(a), upper panel ), the A343D, I340E and S511D mutants did not interact with CaM above detectable levels [6,15,18] and that K526N exhibited a significantly weaker CaM signal than WT (42% lower signal intensity than WT; n = 3, Figure 4(b)). In contrast, the signal for the BFNE mutant R333Q was comparable to that of WT (Figure 4(a,b)).…”

“…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]. Furthermore, CaM plays a crucial role in Kv7.2 channel trafficking [15], therefore regulating polarized axonal surface expression [16,17] and the intracellular transport of Kv7.2 proteins to the plasma membrane [18]. …”

“…It was initially proposed that the reduction in current amplitude is the primary defect leading to disease, with a mere a 25–50% decrease of the M-current being sufficient for pathology [19–21]. Reduction in current levels can be the consequence of a myriad of processes, such as an increase in the degradation rate, reduction in PIP 2 binding, impaired trafficking to the plasma membrane, or dysfunction in the biophysical properties of the channel; all of which have been proposed as responsible for the BFNE phenotype [15,18,22–25]. …”