Abstract
The electrophysiological properties of muscle cells in the
nematode Ascaris suum have been studied extensively (review
DeBell 1965). However, details of the ionic mechanisms
regulating the spontaneous activity of the somatic muscle cell
membrane are still poorly understood. The study described in
this thesis, used the patch-clamp technique to examine ion
channels in the soma membrane of the muscle cells. In addition,
two-electrode voltage-clamp was used to observe the membrane
currents of the muscle cell somata.
The patch-clamp experiments demonstrated the presence of a
high-conductance chloride channel (200pS) spontaneously active
at the resting potential. This channel was voltage sensitive.
When the patch was depolarized the mean open time of the channel
and the probability of opening were both reduced. An additional
feature of this voltage sensitivity was the appearance of
sub-conductance levels when the patch was depolarized.
In patches that contained more than one channel the
proportion of time spent with 1,2,3...N channels open was
analysed in terms of the binominal distribution. The results
indicated that the binomial distribution was not a good
approximation to the data. From this analysis it was concluded
that either the channels in the same patch did not have the same
probability of opening, or that channel openings were not
independent of each other.
Experiments showed that the probability of chloride channel
opening was dependent on the concentration of intracellular
calcium. Increasing the concentration of calcium led to an
increase in the probability of channel opening. The significance
of the calcium sensitivity remains unknown. It was proposed that
these channels were responsible for the high resting
permeability to chloride.
The voltage-clamp experiments demonstrated the presence of
two currents activated by membrane depolarization. When the
muscle cells were bathed in Ringers containing calcium,
depolarization activated an inward current, followed by a large
outward current. The inward current increased in amplitude when
the calciun concentration of the bathing solution was increased,
and was blocked by lanthanizn. The outward current was activated
by steps to +55 mV from a holding potential of -35 mV. This
current had a steep rise and slow decay and was found to be
carried by potassium ions. Depolarizing steps of increased
amplitude, increased the outward current amplitude and decreased
the time to peak of the current. Experiments were carried out to
determine the kinetics of the outward current.
Two blockers of potassiun currents were tried in Ascaris,
these were TEA and 4-AP. TEA [69 mM] was used in the study of
the inward current and blocked most of the outward current. Bath
application of 4-AP [5 mM] blocked a fast-transient component of
the outward current. The current remaining after 4-AP
application had a slow rise time, and a slow decay approximated
by a single exponential, with a time constant of 1.1 s. The 4-AP
resistant current shewed less steady-state inactivation than the
gross outward current. Computer analysis was used to subtract
the 4-AP resistant outward current from the gross outward
current. The subtracted current represented the current blocked
by 4-AP. The decay of the 4-AP blocked current was approximated
by a single exponential, with a time constant of 10.4 ms. The
function of both of these potassium conductances was thought to
be to repolarize the cell after a spike.
