Structure-function analyses of small-conductance, calcium-activated potassium channels

Ion channels are integral membrane proteins present in all cells.
They are highly selective and assure a high rate for ions down
their electrochemical gradient. In particular, small-conductance
calcium-activated potassium channels (SK) are conducting potassium
ions and are activated by binding of calcium ions to calmodulin,
which is constitutively bound to the carboxy-terminus of each SK
channel -subunit. Until now, only three SK channel subunits have
been cloned, SK1, SK2 and SK3. Sequence alignment shows that the
transmembrane and pore regions are highly conserved, while a high
grade of divergence is observed in the amino- and carboxy-termini
of the three subunits. In order to determine the expression of the
different SK channel subtypes, pharmacological tols such as apamin
and d-tubocurarine have been widely used. In this work, I show the
characterization of a novel toxin, tamapin, isolated from the
scorpion Mesobuths tamulus, which targets SK channels. Our
experiments show that this toxin is more potent in blocking SK2
channels than apamin. Furthermore, tamapin only blocked the SK1 and
SK3 channels at higher concentrations, with higher efficiency to
block SK3 than SK1. Therefore, tamapin should be a good
pharmacological tool to determine the molecular composition of
native SK channels underlying calcium-activated potassium currents
in various tissues. Secondly, I determined the molecular mechanism
that prevents the formation of functional SK1 channels cloned from
the rat brain (rSK1). Until now, little information was available
on the rSK1 channels. rSK1 shows highly sequence identity (84%)
with the human homologue, hSK1. hSK1 subunits form functional
potassium channels that are blocked by apamin and d-tubocurarine.
However, when I expressed rSK1 in HEK-293 cells no potassium
currents above background were observed, although
immunofluorescence experiments using a specific antibody against
the rSK1 protein showed expression of the channel. I generated rSK1
core chimeras in which I exchanged the amino-and/carboxy-terminus
with the same region of rSK2 or hSK1. Exchange of amino-and
carboxy-terminus or only of the carboxy-terminus resulted in the
formation of functional potassium channels. Furthermore, I used
these functional chimeras to determine the toxin sensitivity of
rSK1 for apamin and d-tubocurarine. Surprisingly, when these
blockers wre applied, no sensitivity was observed, although hSK1
and rSK1 show a complete sequence identity in the pore region,
which is suggested to contain the binding site for apamin. Finally,
I characterized a novel splice variant of the calcium-activated
potassium channel subunit rSK2, referred to as rSK2-860. The
rSK2-860 cDNA codes for a protein which is 275 amino acids longer
at the amino-terminus when compared with originally cloned rSK2
subunit. Transfection of rSK2-860 in different cell lines resulted
in a surprising expression pattern of the protein. Th protein
formed small clusters around the cell nucleus, but no membrane
stain could be observed. This data shows that the additional 275
amino acid-long stretch at the amino-terminus is responsible for
retention and clustering of rSK2-860 protein. In order to narrow
down the region responsible for this phenotype, I generated
truncated proteins. This resulted in the isolation of an 100 amino
acid-long region that seems to be responsible for the retention and
clustering of rSK2-860 channels. Further truncations and deletions
could help us to find the exact signal which is responsible for
this characteristic behavior of the rSK2-860 protein.