Temperature-Dependent Expression of a Squid Kv1 Channel in Sf9 Cells and Functional Comparison with the Native Delayed Rectifier |
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Authors: | M.W. Brock Z.N. Lebaric H. Neumeister A. DeTomaso W.F. Gilly |
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Affiliation: | (1) Hopkins Marine Station of Stanford University, Department of Biological Sciences, Pacific Grove, CA 93950, USA, US;(2) Neurosciences Program of Stanford University, Stanford, CA 94305, USA, US |
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Abstract: | SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) α-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 (I K) if cultured at the standard postinfection temperature of 27°C. At 18°C, less SqKv1A protein is produced than at 27°C, but cells with I K currents are much more numerous and can survive for at least 20 days postinfection (vs. ∼5 days at 27°C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (∼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 I K 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 I K 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. |
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Keywords: | : Potassium channel — Insect cells — Temperature — Activation — Inactivation — Pharmacology |
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