neurophysiology tutorial papers

• tutorial 1 (Ruppersberg et al, 1990) explores the functional characteristics of heteromultimeric K+ channels
• from wanting to understand the molecular basis of VG K+ channel functional diversity, the rat brain K+ channel forming protein (RCK) was discovered. RCKs form homomultimeric VG K+ channels that have distinct functional characteristics in the Xenopus Laevis
• mRNA coexpression of RKCs in certain regions of the brain means that there is a potential for heteromultimeric VG K+ channels
• in the paper, they look at the differences between homomultimeric RCK1 and RCK4 channels, and heteromultimeric RCK1,4 channels. The preparations used are HeLa cells with cDNA transfection and xenopus oocytes with cRNA transfection
• HeLa cells were transfected with plasmids containing pMSVR1 for RCK1 proteins and pMSVR4 for RCK4 proteins
• in untransfected HeLa cells there was no outwardly rectifying K+ current
• the currents seen passing through RCK1 and RCK4 homomultimeric proteins are different. in RCK4 channels, the currents are transient and insensitive to block by TEA or DTX. in RCK1 channels, there is a noninactivating current that is highly sensitive to TEA with a Kd of 0.6 mM
• Figure 1 shows the traces for RCK1, RCK4 and RCK1,4 in the absence and presence of TEA. All preparations were tested in the presence of 10 mM TEA. RCK1,4 was also tested again in the presence of 100 mM TEA. voltage step was done from -80mV to 0mV for 3.2 seconds. A new cell was used for each type of channel.
• Figure 1 shows that there was no change in the amplitude of the RCK4 channel current (approximately 14nA) In the presence of 10 mM TEA. conversely, the RCK1 current was completely blocked.
• In figure 1, the trace done after washing was to show that the action of TEA was reversible
• in figure 1, the RCK1,4 control for 10 mM TEA had an amplitude of approximately 6 nA. the control amplitude fell to 50 +/- 18% when 10 mM TEA was administered. The RCK1,4 control for 100 mM TEA had an amplitude of approximately 39 nA. TEA presence caused a near complete block. the two HeLa cells in this portion of the experiment caused vastly different responses
• in Figure 1g, HeLa cells were transfected with cDNA for RCK1 and RCK4. the outward K+ current was measured. at a 0 mV depolarising step, the current was initially transient and then non inactivating. the non-inactivating portion of the current was reversible blocked by 10 mM TEA. this was similar to HeLa in just the presence of RCK1. therefore, the blocking of current in mediated by homomultimeric RCK1 channels on both cDNA cotransfection or heteromultimeric that are indistinguishable from RCK1.
• the transient current component of RCK1,4 is pharmacologically different than the RCK4. this was seen in HeLa cells and the xenopus oocytes demonstrated a similar effect. in the HeLa cells, the ransient RCK1,4 current was 50% blocked by 10 mM TEA. with 100 nM DTX, the peak amplification of control reduced to 62%
• HeLa transfection was done using a standard protocol and method by Chen and Okayama. in untransfected HeLa cells, the presence of detectable RCK1 RNA was excluded with northern blot.
• in figure 1, the HeLa cell experiments were conducted at 20 degrees celsius. they were also done 24-48 hours after cDNA transfection. it was a whole cell recording using standard patch clamp techniques. filtered at 120 Hz and sampled at 62.5 Hz. transient recording was corrected for leak and capacitive currents by the P/4 method
• the HEPES buffer (pH = 7.2) was used in both the bathing solution (5 mM) and in the intracellular solution (10 mM) of the HeLa cell experiments
• Figure 2 shows data that was recorded using a cell attached recording method. 10 mM TEA were present for the duration of the experiment to block the non inactivating current
• in figure 2A, depolarising test pulses of 50 ms duration were used. The holding potential was -80 mV. Voltage steps between -50 mV and 40 mV at 10 mV intervals. filtered at 3 kHz and digitised at 4kHz
• Figure 2b shows traces of tail current experiments in order to get an instant I-V relation. Tail currents are due tog gradual closure of channels. responses to voltage steps following activating pulse to 0. voltage steps from -80 mV to 60 mV at 20 mV intervals. there is a linear I-V relationship between -60 and 20 mV for the cotransfected channels. above + 20 mV, saturation is seen. RCK4 I-V relation is super linear
• Figure 2c shows the peak amplitudes of transient current against voltage obtained from measurements in Figure 2a. amplitudes were scaled to give the same slope at -25 mV to allow for comparison. RK1.4 channels begin to saturate at a maximum amplitude of approximately 375 pA whereas RCK4 channels elicit maximum current amplitudes of 1.25 nA
• Figure 2d shows the instantaneous amplitude vs the voltage step. measurements as shown in B but started at -120 mV with 10 mV increments. they have the same slope at -80 mV to allow for comparison. larger instantaneous current in RCK1,4 (approx 0.75 nA) compared with RCK4 (75 pA). some saturation of the RCK 1,4 transient current was seen.
• the differences in saturation are due to differences in open channel characteristics not kinetics. Evaluated whether the properties/ formation of heteromultimeric channels depend on the expression system through whole cell current recordings in HeLa transfected 4 and 1,4. saturation differences were seen again.
• steady state inactivation: responses to test pulses to 0 mV for a 25 ms duration. inactivation curve (co-injected oocytes) steeper and 15 mV shift to more positive potentials than RCK4 channels. half inactivation voltage for RCK4 and RCK1,4 were -58.3 +/- 2 mV and 73.6 +/- 5.2 mV
• the inactivation time constants for RCK4 and RCK1,4 currents were measured in HeLa and oocyte cells. Inactivation time constants for RCK4 and RCK1,4 channels in HeLa cells were 68 +/- 19 ms and 155 +/- 42 ms, respectively. Inactivation time constants for RCK4 and RCK1,4 channels in oocytes were 109 +/- 58 ms and 228 +/- 59 ms, respectively. transient currents in coinjected oocytes recover more than 2 times faster than RCK4.
• Figure 3 was done in cell attached configuration. oocyte was coinjected and a 5 sec conditioning pulse was applied after 180 s pause. paired conditioning and test pulses were made to avoid contributions of test pulse induced inactivation. test pulse duration was 0.5 or 3.2 s. time to 50% recovery was 16.5 +/- 2.5 seconds.
• transient current in cotransfected HeLa was recorded in whole cell patch clamp configuration. 10 mM TEA was applied to ensure there was no non-inactivating current. half time of recovery (from inactivation) for RCK4 and RCK1,4 cells were 7.3 +/- 3.4 s and 2.1 +/- 0.5 s, respectively.
• Figure 4 showed that the single cell channel current of RCK1,4 was similar to that of RCK1 but not RCK4. single cell channel current for RCK 1,4 and RCK1 were 0.67 +/- 0.1 pA and 0.73 +/- 0.02 pA. RCK4 gave a single cell recording of 0.4 +/- 0.06 pA. this was at 0 mV.
• the conclusions reached in this paper showed that heteromultimeric VG Na+ channels have distinctive properties from homomultimeric channels. RCK1 do not inactivate in ms time range, TEA and DTX sensitive. RCK4 has transient current with fast inactivation but insensitive to TEA and DTX. RCK1,4 has current that is transient but sensitive to TEA and DTX. single channel conductance like RCK1 and gating like RCK4
• the paper did not look at in vivo experiments which would be the next approach. could also look at the properties of other RCK channels