Alternative splicing of the human SK3-gene in various muscle cell lines
Summary
The SK3-gene is expressed in various tissues. Especially in skeletal
muscle of patients with myotonic dystrophy there is an elevated expression
of the SK3-gene. Alternative splicing is a process which can generate
multiple transcripts encoding proteins with subtle or opposing functional
differences that can have profound biological consequences. The human
SK3-gene shows four different transcripts. Splice variants containing
exon 4 will increase the length of each P-loop of the outer pore by 15
amino acids. The other alternative splice-site results in a replacement
of exon 1 by exon 1a and codes for a channel with a shorter N-terminal
segemnt. The splice vaiant SK3-ex1 (exon 1 without exon 4) and SK3-ex4
( exon 1 with exon 4) form functional Ca2+-activated and apamin-sensitive
K+ channels when expressed in oocytes. However, they do
have slightly different Ca2+ sensitivities in excised inside-out
patches. The SK3-1a splice variant shows no functionalö expression
and is mainly localized in the cytoplasm. To test whether there is
an age-dependent or disease-dependent difference in the expression pattern
of these transcripts, RT-PCR was made with a variety of muscle cell lines.
We investigated the mouse muscle cell line C2C12 and human muscle cell
lines generated from fetal (F) and adult (A) muscle as well as muscle from
patients with myotonic dystrophy (MD). The splice variant SK3-ex4
was found in F and MD muscle cell lines. The splice variant SK3-1a/ex4
(exon 1a with exon 4) could be detected predominantly in cells of MD- muscle.
Therefore one can expect that some of the immunological reactive SK3 protein
is non functional in the muscle of MD patients. Supported by grants
from the DFG (Gr848/8-2) and the BMBF (iZKF Ulm, B7).
Published as:
Frei E, Wittekindt O, Lehmann-Horn F, Grissmer
S, Jäger H. 2002. Alternative splicing of the human SK3-gene
in various muscle cell lines. European Journal of Physiology Pflügers
Archiv 443:S275
Interaction of cytoplasmic N-tails and C-tails of the small Ca2+ activated potassium channel, KCNN3 (hSK3)
Summary
Functional potassium channels exist only in a tetrameric structure.
From KCNN4 (SK4) it is known that the cytoplasmic C-terminal domain plays
an important role in channel assembly and trafficking (Joiner et al., 2001,
J.Biol.Chem. 276 :37980). KCNN3 channels lacking the N-terminal part of
the channel remain in the cytoplasm, and have a dominant negative effect
on full length isoforms (Wittekindt et al., 2001 Pflügers Archiv 441:R212).
Recently, we were able to show that in yeast two hybrid experiments the
N-terminal part of KCNN3 is interacting with N- and C-tails of KCNN3. The
N-terminal part of KCNN1 and KCNN2 did not show any interaction with N-terminal
parts of KCNN1, KCNN2 or KCNN3. Therefore KCNN3 N- and C-tails seemed to
have a much stronger interaction than the cytoplasmic parts of other KCNN
channels. In order to confirm these experiments that were found in yeast,
we used a prokaryotic expression system and GST-/poly His-tagged constructs
to express the KCNN fusion proteins in the bacterial strain BL21. This
will enable us to verify the results found in yeast with pull-down and/or
immunoprecipitation experiments. Supported by the DFG (Gr848/8-2)
and the BMBF (IZKF Ulm, B7)
Published as:
Frei E, Spindler I, Grissmer S, Jäger H. 2003. Interaction
of cytoplasmic N-tails and C-tails of the small Ca2+ activated
potassium channel, KCNN3 (hSK3). Biophysical
Journal 86:1149pos