Abstract 2327: Redox Regulation of Kv Channels: Obligatory Role of Kv C-terminus in Sensing Pyridine Nucleotide Binding to Kvbeta Subunit
Voltage-gated potassium channels of Kv1 and Kv4 families bind to the ancillary β-subunits that are members of the aldo-keto reductase superfamily which bind pyridine nucleotides with high affinity. Binding of NAD(P)H supports, while the binding of NAD(P)+ abolishes Kvβ1- and Kvβ3-induced inactivation of Kv1.5. The mechanism by which the Kv channel senses the redox state of the pyridine-nucleotide bound to Kvβ remains unknown. To test the possibility that the interaction of Kvα C-terminus with Kvβ affects the differential regulation of Kv currents by oxidized and reduced pyridine nucleotides, we constructed truncation mutations in which 56, 37 or 18 amino acid residues of the Kv1.5 C-terminus were removed and co-expressed with Kvβ3 subunit. When expressed in COS-7 cells, Kvβ3 imparted inactivation to Kv1.5 currents (5.3±1% versus 71±4.1%; control). Inclusion of NADPH (250μM) in the patch-pipette increased the extent and accelerated inactivation of WT Kv1.5-Kvβ3 current (87±1%), whereas NADP+ (1mM) prevented inactivation (11.4±1.5%, p<0.05 versus control). Deletion of 56 C-terminal amino acids of Kv1.5 (Cys547-Leu602, KvΔC56) did not significantly affect Kv expression, its association with Kvβ or Kvβ-mediated inactivation (72.1±2.6%). KvΔC56 did not, however, respond to changes in intracellular pyridine nucleotide concentration when co-expressed with Kvβ3 and neither NAPDH nor NADP+ altered the rate or the extent of inactivation or the voltage-dependence of KvΔC56+Kvβ3 current. Intermediate results were obtained with KvΔC18 and KvΔC38. The glutathione-S-transferase (GST) fusion protein containing peptides from the last 38 (Ile565-Leu602) and 60 (Arg543-Leu602), but not 19 (Asp584-Leu602), amino acids of Kv1.5 C-terminus precipitated Kvβ2 and Kvβ3 in pull-down assays from lysates of transformed bacteria and from mouse brain extracts. The GST-C60 construct did not bind to apoKvβ2, and it displayed higher affinity for Kvβ2:NADPH than for the Kvβ2:NADP+ binary complex. These results suggest that the C-terminal domain of Kv1.5 interacts with Kvβ and that this interaction may be involved in sensing different conformational states of Kvβ bound to either reduced or oxidized pyridine nucleotide.
This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).