Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

12 p.Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A - helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.This work was supported by grants from the VII European framework program managed by the Fondo de Investigaciones Sanitarias (PI071316), from the Spanish Ministry of Education (BFU2009-07581 and SAF2006-1450), the Spanish Ion Channel Initiative Consolider project (CSD2008-00005), and the Basque Government (SAIOTEK SA-2006/00023). A. Alaimo was partially funded by Fundacion Biofisica Bizkaia. PA and JFO held a FPI fellowship from the Spanish Ministry of Science and Innovation (BES-2008-002314). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca2+. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using N-15-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca2+ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca2+ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca2+.This work was supported by grants from the Spanish Ministry of Education (BFU2012-39883 and BFU2009-07581), the Spanish Ion Channel Initiative Consolider project (CSD2008-00005), and the Basque Government (SAIOTEK SA-2006/00023 and 304211ENA9). A. Alaimo and C. Malo were partially funded by Fundacion Biofisica Bizkaia. J. Fernandez-Orth held a FPI fellowship from the Spanish Ministry of Science and Innovation (BES-2008-002314). A. Alberdi holds a JAE-predoctoral CSIC fellowship cofinanced with European Social Funds. G. Bernardo-Seisdedos holds a fellowship from the Basque Country Government (BFI-2011-159). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript