Abstract
Channels are water-filled membrane-spanning proteins, which undergo
conformational changes as they gate, i.e. open or close. These conformational
changes affect both the shape of the channel and the volume of the
water-filled pore. We measured the changes in pore volume associated
with activation, deactivation, C-type inactivation and recovery in
an N-terminal-deleted mutant of the Kv1.4 K$^+$ channel (Kv1.4DeltaN)
expressed in Xenopus oocytes. We used giant-patch and cut-open oocyte
voltage clamp techniques and applied solutes which are too large
to enter the pore mouth to exert osmotic pressure and thus favour
smaller pore volume conformations. Applied intracellular osmotic
pressure (300 mM sucrose) sped inactivation (time constants (tauinactivation):
control, 0.66 +/- 0.09 s; hyperosmotic solution, 0.29 +/- 0.04 s;
n = 5, P < 0.01), sped deactivation (taudeactivation: control, 18.8
+/- 0.94 ms; hyperosmotic solution, 8.01 +/- 1.92 ms; n = 5, P <
0.01), and slowed activation (tauactivation: control, 1.04 +/- 0.05
ms; hyperosmotic solution, 1.96 +/- 0.31 ms; n = 5, P < 0.01). These
effects were reversible and solute independent. We estimated the
pore volume change on inactivation to be about 4500 A3. Osmotic pressure
had no effect when applied extracellularly. These data suggest that
the intracellular side of the pore closes during C-type inactivation
and the volume change is similar to that associated with activation
or deactivation. This is also similar to the pore volume estimated
from the crystal structure of KcsA and MthK K$^+$ channels. Intracellular
osmotic pressure also strongly inhibited re-opening currents associated
with recovery from inactivation, which is consistent with a physical
similarity between the C-type inactivated and resting closed state.
- -u.s.
- 12730347
- algorithms,
- animals,
- blockers,
- calcium
- calcium,
- cardiac,
- cardiovascular,
- channel
- channel,
- channels,
- computer
- conformation,
- electrophysiology,
- female,
- ferrets,
- gating,
- gov't,
- humans,
- ion
- kinetics,
- kv1.4
- l-type,
- models,
- molecular
- molecular,
- myocytes,
- non,
- non-p.h.s.,
- non-u.s.
- oocytes,
- osmotic
- p.h.s.,
- patch-clamp
- porosity,
- potassium
- pressure,
- research
- simulation,
- support,
- techniques,
- u.s.
- voltage-gated,
- water,
- xenopus,
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