Temperature-dependent expression of a squid Kv1 channel in Sf9 cells and functional comparison with the native delayed rectifier.
SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) alpha-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (IK) if cultured at the standard postinfection temperature of 27 degrees C. At 18 degrees C, less SqKv1A protein is produced than at 27 degrees C, but cells with IK currents are much more numerous and can survive for at least 20 days postinfection (vs. approximately 5 days at 27 degrees C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (approximately 3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of IK inactivation in 20 mM external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL IK is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.
Brock
M W
MW
Neurosciences Program of Stanford University, CA 94305, USA.
Lebaric
Z N
ZN
Neumeister
H
H
DeTomaso
A
A
Gilly
W F
WF
D000818:Animals; D001369:Axons / Q000502:physiology*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049832:Decapodiformes; D015151:Immunoblotting; D007700:Kinetics; D051662:Kv1.1 Potassium Channel; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D019789:Tetraethylammonium / Q000494:pharmacology
D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051662:Kv1.1 Potassium Channel; D019789:Tetraethylammonium