Temperature-Dependent Expression of a Squid Kv1 Channel in Sf9 Cells and Functional Comparison with the Native Delayed Rectifier

Abstract

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.

Fast inactivation of Shal (Kv4) K+ channels is regulated by the novel interactor SKIP3 in Drosophila neurons

“…We chose this system because Drosophila proteins are expected to exhibit functional properties in this insect cell line which are more similar to their native function than if expressed in a vertebrate heterologous system (Brock et al 2001). Baculovirus infection of Sf9 cells has indeed proven to be an effective system for analyzing ion channel function and modulation (Brock et al 2001; Gaymard et al 1996; Gubitosi-Klug and Gross 1996; Klaiber et al 1990; Mikhailov et al 1998; Nehrke et al 2000). Recombinant baculovirus particles expressing Shal or SKIP3 were generated, amplified, and used to infect Sf9 cells (see Experimental Methods).…”

Identified Ion Channels in the Squid Nervous System

“…Although the kinetics of inactivation are reasonably similar in Sf9 and GFL cells at positive voltages, inactivation in oocytes is about 2-fold slower. Moreover, inactivation of the native channels clearly follows a biphasic time course at intermediate voltages [105], but this does not appear to be the case in Sf9 cells or oocytes [99]. Probably most important is the fact that in Sf9 cells the rate of inactivation is sensitive to the concentrations of both external K + and TEA ions, consistent with a conventional C-type mechanism [108].…”

“…Reports of Ca 2+ channel expression are encouraging but preliminary. The difficulty of expressing these large polypeptides may be related to the relatively high temperatures at which most expression systems are maintained [99]. This is particularly true for mammalian expression systems, and, to our knowledge no squid channels, including K + channels, have been successfully expressed in mammalian cells.…”

“…Voltage-dependent potassium channel ·-subunits are divided into 4 subfamilies based on sequence homology (K v 1-K v 4 [97,98]), each exhibiting reasonably characteristic functional properties. The squid delayed rectifier begins to open significantly around -40 mV under standard recording conditions [34,99] and displays fast activation kinetics with a pronounced delay. These properties most closely resemble those of the K v 1 subfamily, of which Shaker is a member, and our cloning strategies were made accordingly.…”

“…Comparison of SqK v 1.1A currents in Sf9 cells and Xenopus oocytes with native K + currents in GFL neurons pointed to some clear differences. For example, at 18°C, deactivation kinetics are 10-fold slower in Sf9 cells than in GFL neurons [99] but only about 3-fold slower than in oocytes [102; and unpubl. data].…”