Voltage-dependent Potassium Channel Kv1 Research Articles

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties

The physiological function of ion channels is affected by protein-protein and protein-membrane interactions that modulate their activity and/or localization. Palmitoylation modulates protein function by facilitating targeted membrane association, interaction with other proteins, and determining subcellular localization. In this study, we demonstrate that the voltage-dependent potassium (Kv) channel Kv1.5 is palmitoylated and that the mutation of COOH-terminal cysteines is sufficient to abolish the palmitoylation of the Kv1.5 polypeptide in Chinese hamster ovary (CHO) cells. The labeling represented the thioester linkage of the labeled palmitic acid to cysteine rather than amide and oxygen ester linkages as judged by the release of the palmitic acid upon the treatment of the gel with hydroxylamine at a neutral pH. Site-directed mutagenesis and radiolabeling studies revealed that C593 was the sole site of palmitoylation. The elucidation of the biological function of palmitoylation revealed that the expression of the FLAG-Kv1.5 palmitoylation-deficient mutant (FL-Kv1.5(Palm-)) in stable CHO cells increased membrane expression as determined by the biotinylation of surface proteins and quantitative immunofluorescence analyses of these cells, in turn enhancing the outward potassium current. This enhanced surface expression and the currents were consequential to the slower rate of internalization, causing an increased localization of FL-Kv1.5(Palm-) in the plasma membrane compared with the wild-type FL-Kv1.5 channels. We conclude that the Kv1.5 channel is palmitoylated and that its palmitoylation modulates its biological functions and, therefore, might provide a physiological link between the metabolic state and the expression of Kv1.5 on the plasma membrane.

Manipulation of the Potassium Channel Kv1.1 and Its Effect on Neuronal Excitability in Rat Sensory Neurons

Potassium channels play a critical role in regulating many aspects of action potential (AP) firing. To establish the contribution of the voltage-dependent potassium channel Kv1.1 in regulating excitability, we used the selective blocker dendrotoxin-K (DTX-K) and small interfering RNA (siRNA) targeted to Kv1.1 to determine their effects on AP firing in small-diameter capsaicin-sensitive sensory neurons. A 5-min exposure to 10 nM DTX-K suppressed the total potassium current (I(K)) measured at +40 mV by about 33%. DTX-K produced a twofold increase in the number of APs evoked by a ramp of depolarizing current. Associated with increased firing was a decrease in firing threshold and rheobase. DTX-K did not alter the resting membrane potential or the AP duration. A 48-h treatment with siRNA targeted to Kv1.1 reduced the expression of this channel protein by about 60% as measured in Western blots. After treatment with siRNA, I(K) was no longer sensitive to DTX-K, indicating a loss of functional protein. Similarly, after siRNA treatment exposure to DTX-K had no effect on the number of evoked APs, firing threshold, or rheobase. However, after siRNA treatment, the firing threshold had values similar to those obtained after acute exposure to DTX-K, suggesting that the loss of Kv1.1 plays a critical role in setting this parameter of excitability. These results demonstrate that Kv1.1 plays an important role in limiting AP firing and that siRNA may be a useful approach to establish the role of specific ion channels in the absence of selective antagonists.

The Discovery of Kv1.5 Blockers as a Case Study for the Application of Virtual Screening Approaches.

Different virtual screening techniques are available as alternatives to high throughput screening. These different techniques have been rarely used together on the same target. We had the opportunity to do so in order to discover novel blockers of the voltage-dependent potassium channel Kv1.5, a potential target for the treatment of atrial fibrillation. Our corporate database was searched, using a protein-based pharmacophore, derived from a homology model, as query. As a result, 244 molecules were screened in vitro, 19 of them (7.8%) were found to be active. Five of them, belonging to five different chemical classes, exhibited IC50 values under 10 μM. The performance of this structure-based virtual screening protocol has been compared with those of similarity and ligand-based pharmacophore searches. The analysis of the results supports the conventional wisdom of using as many virtual screening techniques as possible in order to maximize the chance of finding as many chemotypes as possible.

The Discovery of Kv1.5 Blockers as a Case Study for the Application of Virtual Screening Approaches

Different virtual screening techniques are available as alternatives to high throughput screening. These different techniques have been rarely used together on the same target. We had the opportunity to do so in order to discover novel blockers of the voltage-dependent potassium channel Kv1.5, a potential target for the treatment of atrial fibrillation. Our corporate database was searched, using a protein-based pharmacophore, derived from a homology model, as query. As a result, 244 molecules were screened in vitro, 19 of them (7.8%) were found to be active. Five of them, belonging to five different chemical classes, exhibited IC50 values under 10 microM. The performance of this structure-based virtual screening protocol has been compared with those of similarity and ligand-based pharmacophore searches. The analysis of the results supports the conventional wisdom of using as many virtual screening techniques as possible in order to maximize the chance of finding as many chemotypes as possible.

Evidence for phosphorylation-dependent internalization of recombinant human rho1 GABAC receptors.

1. Recombinant wild-type or mutant human rho1 GABA receptors were expressed in human embryonic kidney (HEK) 293 or monkey COS-7 cells and studied using the patch clamp technique. 2. Standard whole-cell recordings with 4 mM Mg-ATP in the patch pipette induced a time-dependent decrease in the GABA-activated current (IGABA) amplitude that was not the result of a decrease in GABA sensitivity. In contrast, IGABA remained stable when recordings were obtained using the perforated patch configuration or with standard whole-cell recording and no Mg-ATP in the patch pipette. 3. The inhibitors of serine/threonine protein kinases KN-62 (20 microM) or staurosporine (20 nM) prevented the time-dependent decrease in the amplitude of IGABA seen in the presence of ATP. Alkaline phosphatase (220 U ml-1), when added to the patch pipette in the absence of ATP, induced a transient potentiation of IGABA. Although the protein kinase C (PKC) activator 4beta-phorbol 12-myristate, 13-acetate (PMA) did not reduce the amplitude of IGABA, inclusion of the catalytic domain of PKC in the recording pipette accelerated the time-dependent decrease in current amplitude. These data suggest that phosphorylation is involved in the regulation of the amplitude of IGABA. 4. Mutation of the three PKC consensus sequences of the rho1 receptor had no significant effect on the decline in IGABA, indicating that direct phosphorylation of these putative sites on the rho1 receptor does not underlie the time-dependent decrease in amplitude. 5. In COS-7 cells transfected with wild-type rho1 receptors, the amplitude of IGABA had completely recovered to the original value when the same cells were repatched after 30-40 min, indicating that the decline in IGABA was a reversible process. 6. The inhibitor of actin filament formation cytochalasin B, when added to the patch pipette in the absence of ATP, induced a time-dependent inactivation suggesting that the actin cytoskeleton may play a role in the regulation of the amplitude. 7. Coincident with the decrease in the amplitude of IGABA, the cell capacitance significantly decreased in the presence of ATP in the patch pipette. This decrease in capacitance was not observed in the absence of Mg-ATP. The decrease in the membrane surface area suggests that receptor internalization could be a potential mechanism for the observed inactivation. 8. At 32 C, compared with 22 C, the rate and magnitude of the decline was increased dramatically. In contrast, at 16 C, no significant change in IGABA was observed over the 20 min recording time. This marked temperature sensitivity is consistent with receptor internalization as a mechanism for the time-dependent decline in IGABA. 9. The specificity of the decrease in IGABA was assessed by coexpressing the voltage-dependent potassium channel Kv1.4 along with the rho1 receptor in HEK293 cells. The amplitude of the potassium current (IKv1.4) exhibited very little decrement in comparison to IGABA suggesting that the putative GABA receptor internalization was not the consequence of a non-specific membrane retrieval.