A novel non-neuronal hSK3 isoform with a dominant-negative effect on hSK3 currents
Summary
We have identified a hSK3-transcript, hSK3_ex1c, which is generated
by alternative splicing. Isoform hSK3_ex1c lacks the cytosolic
N-terminus and the first transmembrane helix and is exclusively expressed
in non-neuronal tissues. hSK3 transfected tsA cells showed
a Ca2+-activated K+ current in patch-clamp experiments,
whereas hSK3_ex1c transfected cells and cells co-transfected with
both isoforms did not. We fused both isoforms to fluorescence proteins
and observed hSK3 localization predominantly in the plasma membrane.
The co-expression of hSK3 + hSK3_ex1c resulted in their cytoplasmic
co-localization. This, hSK3_ex1c has a dominant-negative effect
on hSK3 by preventing its transport into the plasma membrane.
Published as:
Wittekindt OH, Dreker T, Morris.Rosendahl, Lehmann-Horn
F, Grissmer S. 2004. A novel non-neuronal hSK3 isoform with
a dominant-negative effect on hSK3 currents. Cellular Physiology and
Biochemistry 14:23-30.
An apamin and scyllatoxin-insensitive isoform of the human SK3 channel
Summary
We have isolated an hSK3 isoform from a human embryonic cDNA
library that we named hSK3_ex4. This isoform contains a 15 amino
acid insertion within the S5 to P-loop segment. Transcripts encoding hSK3_ex4
are
coexpressed at lower levels with hSK3 in neuronal as well as
non-neuronal tissues. To investigate the pharmacokinetic properties
of hSK3_ex4, we expressed the isoforms hSK3 and hSK3_ex4
in tsA cells. Both isoforms
were similarly activated by cytosolic Ca2+ (hSK3,
EC50 = 0.91 ± 0.4 µM; hSK3_ex4, EC50
= 0.78 ± 0.2 µM) and by 1-ethyl-2-benzimidazolinone (hSK3,
EC50 = 0.17 mM; hSK3_ex4, 0.19 mM). They were both
blocked
by tetraethylammonium (hSK3, Kd = 2.2 mM; hSK3_ex4,
2.6 mM) and showed similar permeabilities relative to K+ for
Cs+ (hSK3, 0.17 ± 0.04, n = 3; hSK3_ex4,
0.17 ± 0.05, n = 3) and Rb+ (hSK3, 0.79 ±
0.04, n =
3; hSK3_ex4, 0.8 ± 0.07, n = 3). Ba2+
blocked both isoforms, and in both cases, the block was strongest at hyperpolarizing
membrane potentials. However, the voltage-dependence of hSK3
was stronger than that of
hSK3_ex4. The most obvious distinguishing feature of this
new isoform was that whereas hSK3 was blocked by apamin (Kd
= 0.8 nM), scyllatoxin (Kd = 2.1 nM), and d-tubocurarine
(Kd = 33.4 µM), hSK3_ex4 was not
affected by apamin up to 100 nM, scyllatoxin up to 500 nM, and d-tubocucarine
up to 500 µM. So far, isoform hSK3_ex4 forms the only
small-conductance calcium-activated potassium (SK) channels, which are
insensitive to
the classic SK blockers.
Published as:
Wittekindt OH, Visan VM, Tomita H, Imtiaz F, Gargus JJ, Lehmann-Horn
F, Grissmer S, Morris-Rosendahl DJ. 2004. An apamin- and scyllatoxin-insensitive
isoform of the human SK3 channel. Molecular
Pharmacology 65:788-801.
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Stephan Grissmer
September 26, 2005