Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism.
The effect of glycosylation on Kv1.l potassium channel function was investigated in mammalian cells stably transfected with Kv1.l or Kv1.1N207Q. Macroscopic current analysis showed that both channels were expressed but Kv1.1N207Q, which was not glycosylated, displayed functional differences compared with wild-type, including slowed activation kinetics, a positively shifted V 1/2, a shallower slope for the conductance versus voltage relationship, slowed C-type inactivation kinetics, and a reduced extent of and recovery from C-type inactivation. Kv1. 1N207Q activation properties were also less sensitive to divalent cations compared with those of Kv1.l. These effects were largely due to the lack of trans-Golgi added sugars, such as galactose and sialic acid, to the N207 carbohydrate tree. No apparent change in ionic current deactivation kinetics was detected inKv1.1N207Q compared with wild-type. Our data, coupled with modelling, suggested that removal of the N207 carbohydrate tree had two major effects. The first effect slowed the concerted channel transition from the last dosed state to the open state without changing the voltage dependence of its kinetics. This effect contributed to the G-V curve depolarization shift and together with the lower sensitivity to divalent cations suggested that the carbohydrate tree and its negatively charged sialic acids affected the negative surface charge density on the channel's extracellular face that was sensed by the activation gating machinery. The second effect reduced a cooperativity factor that slowed the transition from the open state to the dosed state without changing its voltage dependence. This effect accounted for the shallower G-V slope, and contributed to the depolarized G-V shift, and together with the inactivation changes it suggested that the carbohydrate tree also affected channel conformations. Thus N-glycosylation, and particularly terminal sialylation, affected Kv1.l gating properties both by altering the surface potential sensed by the channel's activation gating machinery and by modifying conformational changes regulating cooperative subunit interactions during activation and inactivation. Differences in glycosylation pattern among closely related channels may contribute to their functional differences and affect their physiological roles.
Watanabe
Itaru
I
Department of Biological Sciences, Fordham University, Bronx, NY 10458, USA.
Wang
Hong-Gang
HG
Sutachan
Jhon J
JJ
Zhu
Jing
J
Recio-Pinto
Esperanza
E
Thornhill
William B
WB
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002413:Cations, Divalent / Q000378:metabolism; D006224:Cricetinae; D004553:Electric Conductivity; D045604:Extracellular Fluid / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006706:Homeostasis; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D051662:Kv1.1 Potassium Channel; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats
D002413:Cations, Divalent; D006023:Glycoproteins; D007473:Ion Channels; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051662:Kv1.1 Potassium Channel