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Experimental results from voltage clamp experiments were then translated into changes from these baseline parameters to produce steady state curves for (a) the WT, and (b) the mutation gating parameters at different temperatures.
In this formulation there are only two ion channels — voltage gated-potassium channels, and voltage-gated sodium channels — the latter of which are of particular interest as we aim to parameterise a model that implements the experimentally derived changes in gating parameters m and h for the voltage gated sodium channel to estimate the resultant abnormal dynamics of the neuronal membrane.
α-Hydroxyphenylamide (10 μM) also shifted equilibrium gating parameters of TTX-s Na channels to greater hyperpolarized potentials, slowed recovery from inactivation, accelerated the development of inactivation and exhibited use-dependent block.
Current amplitudes, gating parameters and kinetics were unaffected by either reagents.
Background fluorescence was measured using unlabeled cells and cells labeled with isotype control or secondary antibody alone; and used to set gating parameters between positive and negative cell populations.
All mutants displayed gating parameters (Table 2) similar to those of wild type DmNav1, ruling out the possibility that the alteration of gating behavior was involved in variation of BmK IT2 sensitivity.
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This observation likely relates to interactions between dynamic behaviours of the different voltage gated parameters in the full Hodgkin-Huxley model: through changes in the sodium channel gating parameters, the bifurcation point may be shifted to a location in the combined space that does not allow for bistability.
Since FSC-W provided the maximum size separation on L1210 cells (Table 1), we used L1210 for these experiments and set FSC-W as the first gating parameter.
As shown in Table 1, sorting based on either FSC-H or SSC-H significantly reduced the quality of the size separation for all cell types, as did utilizing SSC-W as the gating parameter.
Equivalent results were obtained when 660/20-A was combined with SSC-A (Figure 4E, right panel, P3, P4 and P5, P6), i.e, rather than showing a two-step separation resulting in four clearly resolved populations, the addition of the 660/20-A autofluorescence parameter did not significantly enhance the separation achieved by the scatter gating parameter alone (see also Figures S3 and S4).
However, the mutation I25C does not affect the gating parameter substantially.
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