Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC314
Characterisation of a chloride conductance in canine chondrocytes
R. Lewis1, W. Wilkinson1, R. Fallman1, R. Whiffin1, R. Barrett-Jolley1
1. Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom.
Healthy chondrocytes can exist with strikingly depolarised resting membrane potentials (RMP) (Wright et al., 1992; Wright et al., 1996). Several papers have investigated the role of potassium conductances in control of RMP (including Wilson et al., 2004) and we showed that the RMP was also dependent upon TRPV5 (Lewis et al., 2011). In the same study we showed that this depolarised RMP was crucial to the control of chondrocyte volume. A number of studies have also suggested that chloride channels are important for control of the RMP (reviewed by Barrett-Jolley et al., 2010) so in this study we investigated the functional expression of these channels in chondrocytes, using both inside-out patch clamp and whole-cell electrophysiology. Chondrocytes were isolated from canine articular cartilage by standard methods (Lewis et al., 2011). Cells were used up to and including the third passage. For inside-out patch experiments, membrane potential (Vm) was calculated as Vm = -Hp-Vj where Hp was the holding potential and Vj the calculated junction potential. Data are expressed as mean ± standard error, p-values are from unpaired t-tests. We identified a population of ion channels with a mean slope unitary conductance of 183±3pS (n = 5) using inside-out patch experiments. These channels reversed at a membrane potential of -34±6mV (n = 5) in the presence of 40mM internal and 158mM external Cl- solutions, indicative of a chloride current (calculated equilibrium potential, ECl ~ -35mV). This channel activity was inhibited by the chloride channel blocker 4-Acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS) at a concentration of 100µM and seen in approximately 30% of patches with a mean open probability (Po) of 0.7±0.1 (n = 3). Application of 100µM SITS decreased channel Po by 83±6% (n = 3; p<0.05). In whole-cell voltage clamp mode, 100µM SITS inhibited voltage ramps by 52±6% (n = 4; p<0.05) at 20mV. We investigated the effect of SITS on the RMP with whole cell current-clamp experiments and found 100µM induced a significant change of +12±3mV (n=5; p<0.01). SITS is a relatively non-selective inhibitor of anionic currents so, to further characterise this chloride current, we used a more specific channel inhibitor; niflumic acid (NFA). NFA inhibits the calcium-activated chloride channel (CaCC), which is also believed to be a volume-sensitive chloride channel. 100µM NFA inhibited whole-cell current by 18±2% (n = 15; p<0.01) at 20mV. Our combined single channel and whole-cell data are consistent with the expression of a mixed population of chloride channels in chondrocytes, including both high conductance maxi-chloride and CaCC-like channels.
Where applicable, experiments conform with Society ethical requirements