Calmodulin regulates the trafficking of KCNQ2 potassium channels

Abstract

Voltage-dependent potassium KCNQ2 (Kv7.2) channels play a prominent role in the control of neuronal excitability. These channels must associate with calmodulin to function correctly and, indeed, a mutation (R353G) that impairs this association provokes the onset of a form of human neonatal epilepsy known as benign familial neonatal convulsions (BFNC). We show here that perturbation of calmodulin binding leads to endoplasmic reticulum (ER) retention of KCNQ2, reducing the number of channels that reach the plasma membrane. Interestingly, elevating the expression of calmodulin in the BFNC mutant partially restores the intracellular distribution of the KCNQ channel. In contrast, overexpression of a Ca(2+)-binding incompetent calmodulin or sequestering of calmodulin promotes the retention of wild-type channels in the ER. Thus, a direct interaction with Ca(2+)-calmodulin appears to be critical for the correct activity of KCNQ2 potassium channels as it controls the channels' exit from the ER.

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

“…To investigate the trafficking of the channels to the plasma membrane, Kv7.3 subunits tagged with an HA epitope at an extracellular loop were used as reporter for surface expression in Xenopus oocytes [15,31,32]. Consistent with the reduction in current amplitude, R333Q mutant surface expression was diminished (~ 50%).…”

“…Remarkably, although the K526N mutant did not diminish current, surface expression was reduced by 40% (Figure 3(a ) ). To confirm the impact of K526N in trafficking in mammalian cells, we fused the AB domain to the membrane protein Tac (interleukin-2 receptor α subunit) and monitored surface expression by flow cytometry in HEK293T cells as previously described [15,33,34]. The results confirmed that this mutation caused a reduction in surface expression (Figure 3(b)).

10.1080/19336950.2018.1511512-F0003Figure 3.Surface expression is reduced in K526N and R333Q mutants.

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“…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)).…”

“…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]. …”