In these experiments barium was used as a charge carrier instead o f calcium for several reasons. Calcium ions are permeable through sodium channels as well as calcium channels, whereas barium is much less permeable through sodium channels. This means that barium currents generated will pass only through calcium channels. Since
the aim o f these experiments was to measure the presence and amplitude o f calcium channel currents, rather than to analyse its inactivation properties, it was not a concern that the barium ions can inhibit the calcium-dependent inactivation o f calcium channels. In fact, this was an additional reason for using barium as a charge carrier as barium currents can be recorded for longer and without the problem o f calcium- dependent inactivation or rundown.
There are many types o f calcium current present in DRG neurones. Calcium currents can be classified according to their voltage sensitivity, in particular, high-voltage activated (HVA) and low-voltage activated (LVA). The high-voltage activated currents are activated at high thresholds (>-20mV) and inactivate at a slow rate (seconds) during a sustained depolarisation. Within this subset o f calcium currents there are components which share similar activation kinetics, such as the L-type and N-type currents. The low-voltage activated current is activated at more negative potentials (—5OmV) and inactivates rapidly. The main component o f the LVA current is the T-type calcium current. The HVA currents can be recorded in isolation from LVA currents in two ways. First by holding the membrane potential level at a depolarised holding potential so that the LVA currents are not activated. Second, by inactivating most o f the LVA channels with a depolarising pulse, followed by a larger depolarising pulse that evokes HVA current whilst the LVA channels are still inactivated. In this study the calcium currents were separated on the basis o f their inactivation properties: the voltage protocol and typical current traces are illustrated in figure 3.16.
HVA type currents were evoked by a command potential from -lOOmV to -lO m V for 50msec and LVA type currents by command potentials from -lOOmV to -5 OmV for 100msec. Figure 3.16b shows that the mean normalised HVA and LVA type calcium channel currents, with barium as the charge carrier o f uninfected and wt HSV 17^ infected DRG neurones, were not significantly different in size.
ininfècted v\t infected
J
InA 20rtis co n tro l H V A L V A 5 - 0 .0 3 E -0.07 J n= 3 4 w t H SV 17' in fected H V A LV A n= 2 4 n=4Figure 3.16. The effect o f wt HSV 17+ infection o f DRG neurones on HVA and LVA type calcium channel currents, a Leak subtracted raw traces illustrating the separation o f LVA and HVA calcium channel currents, h The mean normalised peak HVA and LVA calcium current ± s.e.m o f control or wt HSV 17+ infected DRG neurones for 24 hours. The HVA and LVA type calcium currents were not significantly different between the control and the wt HSV 17+ infected DRG neurones (Student t test, P>0.3).
Not all DRG neurones have a LVA calcium channel current; in fact, only 17% of the wt HSV 17^ infected and 26% of the uninfected DRG neurone population had measurable LVA currents in this experiment.
A family of barium currents was generated with a range of command potentials from - 8OmV to +40mV. The calcium channel current-voltage relationship did not show an obvious shift during a wt HSV 17^ infection, as shown in figure 3.17.
minfected
-80 -20
potential (mV)
5 -0.2 -
■0.4-
-0.6-
Figure 3.17. Calcium current-voltage relationship o f wt HSV 17"^ infected or uninfected DRG neurones, a Two families o f calcium channel currents evoked by a range o f command potentials between -80m V and +40mV from an uninfected and wt HSV 17"^ infected DRG neurone. The current traces shown have had the leak current subtracted, b The graph shows normalised calcium currents ± s.e.m plotted against the command potential from control ( • ) n=35, and wt HSV I?"*" infected (■ ) n=27 DRG neurones.
The activation curves o f the barium currents are shown in figure 3.18. Table 3.6 shows the V5 0, voltage at which the conductance is half maximal, and the slope factor
o f the activation curves for wt HSV 17^ infected and control DRG neurones. There was no significant difference in the electrophysiological characteristics o f the barium conductance (through calcium channels)-voltage relationship o f wt HSV 1Ÿ infected and control DRG neurones.
0.6- g/gma.\ 0 .4 - 0.2- -80 -60 -40 -20 potential (mV)
Figure 3.18. Conductance-voltage relationship for calcium channels o f control or wt HSV IV*" infected DRG neurones. The graph shows the mean normalised conductance ± s.e.m from control ( • ) n= 36, and wt HSV 17"*" infected (■ ) n=27 DRG neurones.
V 5 0 slope factor num ber o f
(mV) (mV) neurones
control -1 6.9 ± 1.1 4.9 ± 0 .5 36 W tH SV 17" -1 4 .7 ± 1.5 4.6 ± 0 .4 27
Table 3.6. Electrophysiological characteristics o f voltage-gated calcium channel currents o f control and wt H SV 17"*" DRG neurones. V5 0 and slope factor values o f control and wt HSV 17"*" infected DRG neurones were not significantly different (Student t test P>0.1).
The inactivation parameter was calculated from the currents evoked by the following protocol; 320msec prepulse potential ranging from -100mV to +15mV, immediately followed by a 320msec command potential to -lOmV, which evoked a maximal current. This voltage protocol and typical traces o f control and wt HSV 17^ infected DRG neurones are shown in figure 3.19a. h^ was calculated by normalising the current generated by the prepulse with the maximal current generated by the test potential. Figure 3.19b illustrates that there is no difference in the steady-state inactivation o f calcium channel currents o f control and wt HSV 17^ infected DRG neurones.
uninfected wtfKV
\r
infectedInA 10ms
0.6 13 J= 0.4 0.2 0.0 - 1 0 0 -80 -60 -40 -20 0 20 prepulse potential (mV)
Figure 3.19. The effect o f prepulse potential on calcium currents from control or wt
HSV infected DRG neurones, a The steady-state inactivation voltage protocol
used to evoke the family o f calcium channel currents for control, uninfected and wt HSV
M'^ infected DRG neurones, b The graph shows normalised calcium currents ± s.e.m plotted against the prepulse potential from control ( • ) n=9, and wt HSV 17"*“ infected (■ ) n=8 DRG neurones. The continuous lines were obtained by fitting a Boltzmann function to the mean normalised data.
V50 slope factor number of (mV) (mV) neurones control -48.2 ± 0.7 14.6 ± 0 .8 9 WtHSV 17" -45.6 ± 0.9 13.2 ± 0 .2 8
Table 3.7. The steady-state inactivation electrophysiological characteristics of calcium channel currents o f uninfected or wt HSV 17"*" infected DRG neurones. There was no significant difference between the V5 0 or slope factor values (Student t test, P>0.6).
In contrast to the sodium currents which were lost 24 hours after infection, the calcium channel currents o f wt HSV 17^ infected DRG neurones remained unchanged in amplitude, activation and steady-state inactivation.