Skip to main content
. Author manuscript; available in PMC: 2015 Apr 19.
Published in final edited form as: Biochem Pharmacol. 2014 Jul 24;91(3):369–379. doi: 10.1016/j.bcp.2014.07.013

Fig. 6.

Fig. 6

Lack of S892 phosphorylation in S892A knock-in mice accelerates fast desensitization of GABAB-activated K+ currents. (A) WT and mutated GB2 alleles. The S892 to alanine mutation (tcc → gcc) and a silent diagnostic NheI restriction site (gctagc) were introduced into exon 19 using homologous recombination in Balb/c embryonic stem (ES) cells. Mutated nucleotides are shown in italic. A neomycin marker (neo) flanked by loxP sites (arrowheads) was used for selection of ES cells. Correctly targeted ES cells (S892A + neo allele) were injected into C57BL/6 blastocysts. A founder mouse was crossed with a Balb/c mouse expressing Cre-recombinase to excise the neomycin cassette, leaving one loxP site behind (S892A allele). The hybridization probe used in the Southern blot in (B) is indicated. A, alanine; N, NheI restriction sites; S, serine. (B) Southern blot of NheI cut genomic DNA from correctly targeted ES cells. The probe labels a 15.3 kb fragment for the WT allele and a 4.3 kb fragment for the S892A + neo allele. (C) Western blot analysis of brain extracts showing that S892A mice express normal levels of GB2, GB1a and GB1b proteins. S892 was phosphorylated in brain extracts of WT but not S892A mice as shown with an antibody specific for phosphorylated S892 (GB2-pS892). Brain extracts of GB2-deficient mice (GB2−/−) [38] confirm the specificity of the GB2 and GB2-pS892 antibodies. β-III-Tubulin was used as a loading control. (D) Representative GABAB-activated K+ currents recorded at −50 mV in response to baclofen application (100 µM) from cultured hippocampal neurons of S892A mice. PKA was activated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; grey trace). Controls represent recordings from untreated neurons (black trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits to the decay phase of K+ currents during baclofen application (enlarged on the right). (E) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization in WT and S892A neurons. Data are means ± SD, n = 5–7. *,p < 0.05; ***, p < 0.001; Sidak’s multiple comparison test.