Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation
Section snippets
Antibody production
The regions corresponding to amino acids M1-L77 of KCNQ2 (to produce the αKN2 antisera), S617-Q676 of KCNQ3 (αKC3) and M1-R88 of KCNQ5 (αKN5) were amplified by PCR and subcloned into pThioHis (Invitrogen), pRSET (Invitrogen) and pGEX3X (Pharmacia), respectively. The E. coli strain, BL21.De3(pLys) (Stratagene, La Jolla, CA, USA), was transformed and fusion proteins were purified according to the manufacturer's instructions with the following columns: HisBind (Novagen, Madison, WI, USA) for the
Antisera characterization
We have determined the specificity of the antisera we generated by using membrane extracts of HEK293T cells transiently expressing human KCNQ2 subunits, and from HEK293 cells stably expressing human KCNQ3, KCNQ4 or KCNQ5. Each antisera recognized a band close to the theoretical MW of KCNQ from the membranes expressing the corresponding subunit, but not from membranes prepared from cells expressing other subunits or non-transfected cells (Fig. 1A).
- C
cortex lysate
- CA1–4
hippocampal CA fields
- DG
Abbreviations used in the figures
Discussion
In the present study, we have generated an antiserum against the KCNQ5 protein that has permitted us to analyze aspects of its biochemical properties and its distribution in brain tissue. In contrast to human brain tissue (Cooper et al., 2000), we found that KCNQ2 and KCNQ3 subunits from rat brain tissue were easily solubilized with Triton X-100. The basis of this differential behavior is unclear, perhaps reflecting species differences in the interaction with cytoskeletal components (Cooper et
Acknowledgements
This work was supported by the EU grant QLGT-1999-00827, a grant from the ‘Fondo de Investigaciones Sanitarias' FIS01/1136, Comunidad de Madrid 08.5/0011/2001.1 and the ‘Ministerio de Ciencia y Tecnología' SAF2000-0159. We thank Drs. Thomas Jentsch and Bjoern Schroeder for providing the KCNQ5 clone prior to its publication, and Dr. Mark Sefton for critical comments and editorial assistance.
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2016, European Journal of PharmacologyCitation Excerpt :The KCNQ subfamily of potassium channels is formed by five member subunits, named KCNQ1 (Kv7.1)–KCNQ5 (Kv7.5). KCNQ1 is mainly expressed in the heart, KCNQ2 and KCNQ3 are restricted to the nervous system and KCNQ4 and KCNQ5 are more widely expressed (Jentsch, 2000; Shah et al., 2002; Yus-Najera et al., 2003). All the KCNQ subunits can form homo-tetrameric channels, but the KCNQ3 subunit also forms hetero-tetramers with either KCNQ2 or KCNQ5 subunits (Jentsch, 2000).
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2015, Experimental Eye ResearchCitation Excerpt :A possible relationship between KCNQ5 and GFAP in the rat retina has been sustained by the presence of potassium channels, potassium currents and GFAP expression in astrocytes or Müller cells in the retina (Bay and Butt, 2012; Chavira-Suárez et al., 2011; Connors and Kofuji, 2006; Zhao et al., 2012). KCNQ5 currents have been reported in monkey retina (Pattnaik and Hughes, 2012) and KCNQ5 expression has been described in brain astrocytes (Yus-Nájera et al., 2003). Many glial cell functions have also been associated with potassium channels, such as regulation of cell proliferative activity (Newman, 2004; 2003), performance of retinal K+ homeostasis through inwardly rectifying potassium (Kir) channels (Raap et al., 2002), and cell-to-cell communication (Bay and Butt, 2012).
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2014, Molecular and Cellular NeuroscienceCitation Excerpt :Our data are thus consistent with these observations and point that the KCNQ3 subunit orchestrates the heteromeric interaction. This may be especially important, as suggested by the observations that KCNQ3 is the most promiscuous KCNQ subunit, assembling with KCNQ2 (Roche et al., 2002; Shapiro et al., 2000; Wang et al., 1998), KCNQ5 (Lerche et al., 2000; Schroeder et al., 2000; Yus-Najera et al., 2003), and also KCNQ4 in heterologous systems (Bal et al., 2008; Kubisch et al., 1999). We thus suspect that CaM is an auxiliary subunit which promotes the multimerization of KCNQ3 with KCNQ2 (Fig. 7).
- 1
These authors contributed equally to this work.