Evaluation of functional interaction between K(+) channel alpha- and beta-subunits and putative inactivation gating by Co-expression in Xenopus laevis oocytes

Abstract

Animal K(+) channel alpha- (pore-forming) subunits form native proteins by association with beta-subunits, which are thought to affect channel function by modifying electrophysiological parameters of currents (often by inducing fast inactivation) or by stabilizing the protein complex. We evaluated the functional association of KAT1, a plant K(+) channel alpha-subunit, and KAB1 (a putative homolog of animal K(+) channel beta-subunits) by co-expression in Xenopus laevis oocytes. Oocytes expressing KAT1 displayed inward-rectifying, non-inactivating K(+) currents that were similar in magnitude to those reported in prior studies. K(+) currents recorded from oocytes expressing both KAT1 and KAB1 had similar gating kinetics. However, co-expression resulted in greater total current, consistent with the possibility that KAB1 is a beta-subunit that stabilizes and therefore enhances surface expression of K(+) channel protein complexes formed by alpha-subunits such as KAT1. K(+) channel protein complexes formed by alpha-subunits such as KAT1 that undergo (voltage-dependent) inactivation do so by means of a "ball and chain" mechanism; the ball portion of the protein complex (which can be formed by the N terminus of either an alpha- or beta-subunit) occludes the channel pore. KAT1 was co-expressed in oocytes with an animal K(+) channel alpha-subunit (hKv1.4) known to contain the N-terminal ball and chain. Inward currents through heteromeric hKv1. 4:KAT1 channels did undergo typical voltage-dependent inactivation. These results suggest that inward currents through K(+) channel proteins formed at least in part by KAT1 polypeptides are capable of inactivation, but the structural component facilitating inactivation is not present when channel complexes are formed by either KAT1 or KAB1 in the absence of additional subunits.

Figure 1

Analysis of KAB1 co-expression effects on KAT1 currents. Time-dependent K⁺ currents are shown for oocytes injected with approximately 10 μg of KAT1 cRNA (A), approximately 10 μg each of KAT1 and KAB1 cRNA (B), or water (C). During recordings shown in A, B, and C, the holding potential was −60 mV, the current was recorded at −160-, −120-, −80-, −40-, 0-, and +40-mV step voltages, and the current and time axes are indicated by vertical and horizontal bars, respectively. D, K⁺ currents (±se) recorded from oocytes (n ≥ 13 for each of the treatments shown) maintained at a holding potential of −60 mV during a step voltage to −160 mV. Pooled data are presented for recordings of K⁺ currents from oocytes injected with either KAB1 (alone), KAT1 (alone), or KAT1 and KAB1 (together) cRNA. E, G/G_max at varying command potentials for K⁺ currrents recorded from oocytes injected with KAT1 cRNA or with KAT1 and KAB1 cRNA. In many cases, the data points for the two treatments overlap; only one data point is visible at many of the step voltages. F, Time-dependent currents (−60-mV holding potential, −160-mV step voltage). Data for KAT1 alone and in the presence of KAB1 are shown, along with a superimposition of the two currents by adjusting the KAT1 data.

Figure 2

A, Relationship between the amount of KAT1 cRNA injected into oocytes and current amplitude. Means (±se; n ≥ 13) of currents recorded at a −160-mV step voltage (−60-mV holding potential) are shown for oocytes injected with the standard (approximately 10 ng) amount of cRNA and also for oocytes injected with RNA prepared at either a 10-fold higher concentration (designated as level 10) or a 10-fold dilution (designated as level 0.1). The standard level of cRNA (level 1) was chosen because this treatment typically results in maximal currents of approximately 2 μA at a step voltage of −160 mV. B, Immunoblot analysis of KAB1 protein expressed in oocytes. Three oocytes were dissolved in SDS-PAGE sample buffer after injection of either approximately 10 ng (lane 1), approximately 50 ng (lane 2), or no KAB1 cRNA (lane 3, control oocytes injected with water).

Figure 3

Time-dependent K⁺ currents recorded from oocytes injected with cRNA generated from various KAT1 constructs. In this experiment, the holding potential was −40 mV, and currents were recorded at step voltages ranging from −60 to −180 mV in 20-mV increments on oocytes injected with (10 ng) KAT1 (A), KAT1^Δ1–28 (B), or NAB-KAT1^Δ1–28 (C) cRNA. In all cases, currents were recorded from a minimum of 10 oocytes and, for a given treatment, reproducible results were obtained from at least two independent experiments (i.e. two batches of oocytes). Currents shown are representative of all recordings for a particular treatment.

Figure 4

Time-dependent K⁺ currents recorded from oocytes injected with hKv1.4 (10 ng) and/or various constructs of KAT1 cRNA (10 ng). Treatments (i.e. cRNA) were as follows: hKv1.4 (A), KAT1^Δ1–28 (B), hKv1.4 and KAT1^Δ1–28 (C and D), and hKv1.4 and NAB-KAT1^Δ1–28 (E and F). For A, C, and E, the voltage-clamp protocol generated outward currents, the holding potential was −70 mV, and the step voltage was varied between −80 and +40 mV in 20-mV increments. For B, D, and F, the voltage-clamp protocol generated inward currents, the holding potential was −40 mV, and the step voltage was varied between −60 and −180 mV in 20-mV increments. In all cases, currents were recorded from a minimum of 10 oocytes and, for a given treatment, reproducible results were obtained from at least two independent experiments (i.e. two batches of oocytes). Curents shown are representative of all recordings for a particular treatment.