Modulation of the rat potassium channel Kv1.5

Daniel Benjamin Saal, Yale University.

This is an Open Access Thesis


1. Kv1.5 is a delayed rectifier-type potassium channel. We have examined the localization of the channel protein using a polyclonal antibody against Kv1.5. This channel is expressed in numerous regions of the brain, primarily in glial cells. Its expression is particularly intense in the endfoot processes which abut the microvasculature of the brain. It is also expressed in the cell bodies and cellular processes in the pituitary.2. Immunoprecipitation of the Kv1.5 protein, from HEK 293 cells which were transformed with the Kv1.5 gene, revealed that the channel is phosphorylated and that treatment of cells with dibutyryl cyclic AMP (dibcAMP) or dibutyryl cyclic GMP (DIB cGMP) causes an increase in the level of Kv1.5 channel phosphorylation. There are three C-terminal PKA consensus phosphorylation sites in rat Kv1.5. Removal of these three sites collectively eliminates this phosphorylation. When these sites are mutated separately, however, the channel still is phosphorylated in response to activators of PKA. This indicates that multiple sites in the Kv1.5 protein are phosphorylated by PKA.3. Phosphorylation of Kv1.5 is long lasting. The cAMP-induced phosphorylation remains for over an hour after the removal of the kinase activator.4. The Kv1.5 phosphorylated protein is sensitive to deglycosylation by ENDO H which indicates that phosphorylation takes place in the Golgi or the endoplasmic reticulum prior to its transport to the plasma membrane.5. We have studied the currents produced by Kv1.5 when it is transfected into HEK 293 cells. Currents recorded using the whole cell technique activate rapidly and undergo little inactivation during depolarizing pulses. Mutation of the first and third C-terminal PKA consensus phosphorylation sites shifts the conductance voltage curves in the negative direction relative to those derived from wild type currents. Treatment with H8, an inhibitor of protein kinases A and G, also shifts the conductance voltage curve of wild type Kv1.5 in the negative direction.6. I$\sb{\rm Kv1.5}$ currents recorded using the nystatin perforated patch technique inactivate with mean time constants of 49 $\pm$ 16 msec. Simultaneous removal of the first and third C-terminal sites by site directed mutagenesis slows or eliminates this inactivation. Treatment of cells expressing the wild type Kv1.5 channel with H8 also slows the rate of inactivation.