Journal of Neuroscience, Vol 8, 4765-4779, Copyright © 1988 by Society for Neuroscience
Single-channel analysis of four distinct classes of potassium channels in Drosophila muscle
WN Zagotta, MS Brainard and RW Aldrich
Department of Neurobiology, Stanford University School of Medicine, California 94305.
A number of mutations have been shown to affect potassium channels in
Drosophila muscle. Single-channel analysis of the effects of mutations will
prove a powerful approach for studying the molecular mechanisms of ion
channel gating. As an initial step towards studying the effects of
mutations at the single-channel level, we have characterized wild-type
potassium channels in cultured embryonic myotubes using whole-cell,
cell-attached, inside-out, and outside-out configurations of the patch-
clamp technique. The myotubes differentiate in vitro from primary cultures
of late-gastrula stage embryos of Drosophila. The whole-cell outward
currents develop in a characteristic sequence. At 8 hr after plating a
small delayed outward current is present. Between 10 and 12 hr after
plating an A-type outward current develops, followed, between 13 and 16 hr,
by a large increase in the delayed current. The A-type current is absent at
all developmental stages in myotubes homozygous for the mutant ShKS133. At
least 4 different types of potassium channels contribute to the whole-cell
outward currents: a fast transient 14 pS A-type potassium channel (A1), a
slowly inactivating 14 pS potassium channel (KD), a 40 pS potassium channel
that does not inactivate during voltage pulses up to 2.4 sec in duration
(KO), and a 90 pS potassium channel that is strongly activated by membrane
stretch (KST). Channels indistinguishable from the KD and KST channels were
also observed in patch-clamp studies on larval body wall muscle fibers. A1
channels were also present in intact dorsal longitudinal flight muscles.
The A1 channel underlies the rapidly inactivating component of the
whole-cell current. It inactivates with a similar time course and voltage
dependence to the A-current and is similarly blocked by 5 mM 4-
aminopyridine. The KD channel underlies a large fraction of the delayed
component of the whole-cell current. Ensemble averages of single KD
channels inactivate with the same time course as the delayed current. The
KO channel represents a smaller fraction of the whole-cell delayed outward
current. Its increase in open probability with voltage is due primarily to
a voltage dependence of its closed times. The KST channel is voltage and
calcium independent and would therefore only contribute to the leak
whole-cell current.
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