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

Abstract

Heteromers of Kv7.2/Kv7.3 subunits constitute the main substrate of the neuronal M-current that limits neuronal hyper-excitability and firing frequency. Calmodulin (CaM) binding is essential for surface expression of Kv7 channels, and disruption of this interaction leads to diseases ranging from mild epilepsy to early onset encephalopathy. In this study, we addressed the impact of a charge neutralizing mutation located at the periphery of helix B (K526N). We found that, CaM binding and surface expression was impaired, although current amplitude was not altered. Currents were reduced at a faster rate after activation of a voltage-dependent phosphatase, suggesting that phosphatidylinositol-4,5-bisphosphate (PIP2) binding was weaker. In contrast, a charge neutralizing mutation located at the periphery of helix A (R333Q) did not affect CaM binding, but impaired trafficking and led to a reduction in current amplitude. Taken together, these results suggest that disruption of CaM-dependent or CaM-independent trafficking of Kv7.2/Kv7.3 channels can lead to pathology regardless of the consequences on the macroscopic ionic flow through the channel.

The S2–S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation

“…CaM was reported to be an auxiliary subunit of Kv7 channels (Wen and Levitan, 2002). Disrupted CaM interactions with Kv7 channels have been shown to be tightly associated with severely impaired Kv7 surface expression (Ghosh et al, 2006;Shamgar et al, 2006;Etxeberria et al, 2008;Alaimo et al, 2009Alaimo et al, , 2018Liu and Devaux, 2014;Chang et al, 2018), illustrating the vital role of CaM in Kv7 assembly and trafficking, which is also supported by our study. Additionally, the stoichiometry for CaM binding to the Kv7 subunit was 1:1 (Sun andMacKinnon, 2017), andBernardo-Seisdedos et al (2018) reported that the Kv7.2 A and B helices could not be purified in the absence of CaM and that the complex could not be dissociated at any calcium concentration in vitro.…”

“…Earlier studies mostly focused on the role of the C-terminal domain of Kv7 channels in CaM binding and CaM regulation. Mutations in the C-terminal domain impairing CaM binding affect channel assembly, trafficking, and gating (Ghosh et al, 2006;Shamgar et al, 2006;Etxeberria et al, 2008;Alaimo et al, 2009Alaimo et al, , 2018Liu and Devaux, 2014;Tobelaim et al, 2017;Chang et al, 2018). Here, our data provided new insight into CaM regulation of Kv7 channels by proving that the S2-S3 loop of Kv7.4 channels is essential for CaM-mediated calcium-dependent regulation of Kv7.4 activation.…”

“…The Kv7 A and B helices, which are located in the cytoplasmic C-terminal domain and close to the pore, are identified as CaM binding sites (Wen and Levitan, 2002;Yus-Najera et al, 2002;Sachyani et al, 2014;Strulovich et al, 2016;Sun and MacKinnon, 2017;Chang et al, 2018). Disrupted CaM interactions with Kv7.1 (Ghosh et al, 2006;Shamgar et al, 2006), Kv7.2 (Etxeberria et al, 2008;Alaimo et al, 2009Alaimo et al, , 2018, heteromeric Kv7.2/Kv7.3 (Liu and Devaux, 2014), or Kv7.4 (Chang et al, 2018) have been shown to be tightly associated with severely impaired Kv7 surface expression, which illustrates the vital role of CaM in Kv7 assembly and trafficking. Numerous studies about the effects of calcium on the interaction between CaM and Kv7 revealed that both Ca 2+ /CaM and Apo/ CaM forms bind to the channels (Wen and Levitan, 2002;Yus-Najera et al, 2002;Ghosh et al, 2006;Shamgar et al, 2006;Bal et al, 2008), whereas details of the structure of the Kv7:CaM complex are calcium dependent.…”

The Crossroad of Ion Channels and Calmodulin in Disease

“…There are contrasting reports regarding the effect of the K526N (K554N in isoform 1) mutation on CaM binding, voltage-dependence, and membrane surface expression (Table 4; Figure 3). There is general agreement on an impact on PIP 2 sensitivity and a tendency to increase current density [105,106,113]. Similar to other mutations at or close to the CaM binding surface, channels carrying this mutation are less efficient at controlling neuronal excitability [106].…”

“…Whereas the R333W mutant interferes with CaM binding, the R333Q mutation does not. However, both mutations affect channel trafficking [105,106] (Table 4; Figure 3).…”