| pA2
online © Copyright 2003 The British Pharmacological Society |
051P
University
of Manchester Autumn Meeting September 2003 |
|
|
 Print Abstract Search PubMed
for: |
The 5-HT3 receptor is a pentameric, non-selective cation channel of the transmitter-gated ion channel (TGIC) receptor superfamily. To date, two 5-HT3 subunits have been identified: 5-HT3A and 5-HT3B. In recombinant expression systems, human homomeric 5-HT3A receptors mediate an inwardly rectifying cation conductance, whilst in cells expressing human heteromeric 5-HT3A+5-HT3B receptors, the inward rectification of cation conductance is abolished (Davies et al., 1999). In a recent study, the single channel conductance of human 5-HT3 receptors was shown to be markedly influenced by arginine residues within a putative amphiphilic helix (the 'HA stretch') located in the M3-M4 cytoplasmic loop (Kelley et al., 2003). It was hypothesised that these residues might also influence inward rectification in 5-HT3 receptors. A triple point mutation ('ATR') of the 5-HT3A subunit was constructed, whereby three arginine residues (R432Q, R436D, R440A) were mutated to the aligned human 5-HT3B subunit residues. cDNAs were transfected by electroporation and expressed in tsA-201 cells as described by Kelley et al., (2003).Control cells were transfected with either wildtype 5-HT3A or 5-HT3A+5-HT3B cDNA. Current-voltage (I-V) relationships of 5-HT (10 µM) induced whole cell currents were determined by a voltage ramp protocol (-100 mV to +40 mV) (Brown et al., 1998). Chord conductance (Gc) was calculated at +40 mV and -100 mV and the ratio (Gc(+40)/Gc(-100)) used as a measure of rectification, with a value of unity indicating the absence of rectification. Reversal potentials (E5-HT) were also determined.
As summarised in Table 1, there was a significant effect of the receptor construct on chord conductance ratios confirmed by a one-way analysis of variance [F(2,9)=6.12, p<0.05], whilst E5-HT was not significantly influenced [F(2,9)=1.99, p>0.05].
|
Gc
ratios and E5-HT of wildtype and
mutant 5-HT3 receptors
|
||
|
Receptor
|
Gc(+40)/Gc(-100)
|
E5-HT(mV)
|
|
5-HT3A
|
0.3
± 0.01†
|
-1.3
± 0.95
|
|
5-HT3A+5-HT3B
|
0.67± 0.07*
|
-4.4 ± 1.6
|
|
ATR
|
0.63
± 0.09*
|
-3.4
± 0.77
|
Values
are mean + s.e.m., n=3-4 cells. * significantly different from
5-HT3A, † significantly different from
5-HT3A+5-HT3B,
Tukey's t, p<0.05
For the ATR constructs, reduction of intracellular [Cl-] to 15 mM by partial replacement of KCl by K gluconate within the pipette solution, produced no significant difference in E5-HT (0.32 ± 2.6 mV, n = 3) and the Gc(+40)/Gc(-100) ratio remained significantly greater (0.82 ± 0.18, n = 3, p < 0.05) than that of wildtype 5-HT3A receptors. The present study suggests that charged residues within the large M3-M4 cytoplasmic loop influence ion permeation and that positively charged residues may act as a filtering mechanism to impede outward cation conductance in 5-HT3 receptors.
Brown,
A.M. et al.,(1998) J Physiol 507, 653-65.
Davies, P.A. et al., (1999) Nature 397, 359-63.
Kelley, S.P. et al., (2003) Nature. In press.