Transient voltage-gated potassium channels in cultured hippocampal astrocytes
In the nervous system, the roles of Kv channels are well established as being critical for regulating action potential frequency, membrane potential, and neurotransmitter release. However, their role in glial cells, a non-excitable cell type, is yet to be fully understood. Whole-cell current kinetics, pharmacology, immunocytochemistry and RT-PCR were used to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ~70%, 10% and less than 5% of total A current is associated with Kv4, Kv3 and Kv1 channels, respectively. In addition, pharmacology and kinetics provide novel evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Since astrocytes are known to be subjected to neuronal firing frequencies of up to 200 Hz in the hippocampus, the role of A currents in membrane voltage oscillations was assessed. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. Astrocytes and HEK293 cells were used to investigate the mechanism of the previously found GABAA induced anion-mediated reduction of Kv channels in more detail. Astrocytes demonstrate an anion concentration specific depolarizing effect on inactivating A-type (also termed transient voltage-gated) K⁺ channel activation kinetics whereas a hyperpolarizing effect was seen upon expression of Kv4.2 or Kv1.4 in HEK293 cells, but only after disruption of the cytoskeleton using cytochalasin D. It is hypothesized that cytoskeletal interactions and Cl⁻ -mediated effects are mediated through N-terminal conformational stabilities. In summary, the results indicate that hippocampal astrocytes in vitro express multiple A type Kv channel α-subunits with accessory, Ca²⁺ -sensitive cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Functions of these channels remain to be determined in a physiological setting, but suggest that A-type Kv channels enable astrocytes to respond rapidly with membrane voltage oscillations to high frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity. Furthermore, studies of anion and cytoskeletal effects on Kv channels demonstrate channel function to be highly localized/targeted and susceptible to changes in ionic environment.
cytoskeleton, Kv channel, HEK293, localization
Doctor of Philosophy (Ph.D.)