Elsevier

Neuroscience

Volume 120, Issue 2, 22 August 2003, Pages 353-364
Neuroscience

Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation

https://doi.org/10.1016/S0306-4522(03)00321-XGet rights and content

Abstract

The KCNQ family of voltage-dependent non-inactivating K+ channels is composed of five members, four of which (KCNQ2–5) are expressed in the CNS and are responsible for the M-current. Mutations in either KCNQ2 or KCNQ3 lead to a hereditary form of dominant generalized epilepsy. Using specific antisera to the KCNQ2, KCNQ3 and KCNQ5 subunits, we found that KCNQ3 co-immunoprecipitated with KCNQ2 and KCNQ5 subunits, but no association was detected between KCNQ2 and KCNQ5. Intense KCNQ5 immunoreactivity was found to be widely distributed throughout the temporal neocortex and the hippocampal formation. In these structures, both pyramidal and non-pyramidal neurons and a population of glial cells in the white matter expressed the KCNQ5 subunit. In the sclerotic areas of the CA fields of epileptic patients, a marked loss of KCNQ5 immunoreactive pyramidal neurons was found in relation with the loss of neurons in these regions. However, in the regions adjacent to the sclerotic areas, the distribution and intensity of KCNQ5 immunostaining was apparently normal. The widespread distribution of KCNQ5 subunits, its persistence in pharmacoresistant epilepsy, along with the significant role of the M-current in the control of neuronal excitability, makes this protein a possible target for the development of anticonvulsant drugs.

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).

    Abbreviations used in the figures

    C

    cortex lysate

    CA1–4

    hippocampal CA fields

    DG

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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