Inactivation of Kv3.3 potassium channels in heterologous expression systems.
Kv3.3 K+ channels are believed to incorporate an NH2-terminal domain to produce an intermediate rate of inactivation relative to the fast inactivating K+ channels Kv3.4 and Kv1.4. The rate of Kv3.3 inactivation has, however, been difficult to establish given problems in obtaining consistent rates of inactivation in expression systems. This study characterized the properties of AptKv3.3, the teleost homologue of Kv3.3, when expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. We show that the properties of AptKv3.3 differ significantly between CHO and HEK cells, with the largest difference occurring in the rate and voltage dependence of inactivation. While AptKv3.3 in CHO cells showed a fast and voltage-dependent rate of inactivation consistent with N-type inactivation, currents in HEK cells showed rates of inactivation that were voltage-independent and more consistent with a slower C-type inactivation. Examination of the mRNA sequence revealed that the first methionine start site had a weak Kozak consensus sequence, suggesting that the lack of inactivation in HEK cells could be due to translation at a second methionine start site downstream of the NH2-terminal coding region. Mutating the nucleotide sequence surrounding the first methionine start site to one more closely resembling a Kozak consensus sequence produced currents that inactivated with a fast and voltage-dependent rate of inactivation in both CHO and HEK cells. These results indicate that under the appropriate conditions Kv3.3 channels can exhibit fast and reliable inactivation that approaches that more typically expected of "A"-type K+ currents.
Fernandez
Fernando R
FR
Neuroscience Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Morales
Ezequiel
E
Rashid
Asim J
AJ
Dunn
Robert J
RJ
Turner
Ray W
RW
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D017434:Protein Structure, Tertiary; D051381:Rats; D051670:Shaw Potassium Channels
D018076:DNA, Complementary; C085724:Kcnc3 protein, mouse; C484184:Kcnc3 protein, rat; D008164:Luminescent Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051670:Shaw Potassium Channels; D049452:Green Fluorescent Proteins; D011188:Potassium