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Emerging Concepts in Ion Channel Biophysics
Wednesday Speaker Abstracts
22
Characterization of Dynamic Kv Channel-toxin Structures with Voltage Clamp
Spectroscopy
Sebastian Fletcher-Taylor
1
, Parashar Thapa
2
, Jon T. Sack
2
,
Bruce E. Cohen
1
.
1
Lawrence Berkeley National Laboratory, Berkeley, CA, USA,
2
University of California Davis,
Davis, CA, USA.
Ion channels are polymorphic membrane proteins whose states and transitions have been
identified by electrophysiology, and whose static structures have begun to yield to X-ray and EM
techniques. These structures have offered images of individual states, giving us starting points
for identifying the complex and transient structural changes that give rise to channel physiology.
To understand the structural changes that underlie the gating of voltage-gated K
+
channels, we
have synthesized fluorescent channel activity probes based on the tarantula toxin guangxitoxin-
1E (GxTX), and used these to image structural changes in the GxTX-Kv complex. We have
synthesized chemoselective point mutants of GxTX, an inhibitory cystine knot peptide that binds
selectively to Kv2 channels, and labeled them with a novel environment-sensitive far-red
fluorophore, JP, whose emission is sensitive to the polarity of its surroundings. JP-GxTX
fluorescence measured in live cell membranes is dependent on the labeling site on the toxin and
changes in response to Kv2 voltage activation. Using spectral images from patch clamped cells
(or
Voltage Clamp Spectroscopy
, VCS) we have developed curve fitting techniques constrained
by regression analysis to identify structural changes of the complex, and to reveal the probability
of Kv2 ion channel activation at each voltage. Emission spectra of the JP27 GxTX mutant
comprise at least 2 species whose emission peaks and amplitudes vary reversibly with membrane
potential. Depolarization redshifts the emission peaks of both species, suggestive of numerous
channel-toxin structures and transitions to more polar environments during activation gating.
These voltage-dependent spectra offer unprecedented detail of the dynamic structural changes of
a channel-toxin complex in live cells. We expect VCS to reveal ion channel activity with greater
structural and temporal resolution than methods relying on integrated and broadband
fluorescence signals.
Calcium, Calmodulin, and Potassium Channels
Richard Aldrich
.
University of Texas, Austin, USA.
No Abstract