Heteromultimeric potassium channels formed by members of the Kv2 subfamily

Abstract

Four alpha-subunits are thought to coassemble and form a voltage-dependent potassium (Kv) channel. Kv alpha-subunits belong to one of four major subfamilies (Kv1, Kv2, Kv3, Kv4). Within a subfamily up to eight different genetic isotypes exist (e.g., Kv1.1, Kv1.2). Different isotypes within the Kv1 or Kv3 subfamily coassemble. It is not known, however, whether the only two members of the vertebrate Kv2 subfamily identified thus far, Kv2.1 and Kv2.2, heteromultimerize. This might account for the lack of detection of heteromultimeric Kv2 channels in situ despite the coexpression of Kv2.1 and Kv2.2 mRNAs within the same cell. To probe whether Kv2 isotypes can form heteromultimers, we developed a dominant-negative mutant Kv2.2 subunit to act as a molecular poison of Kv2 subunit-containing channels. The dominant-negative Kv2.2 suppresses formation of functional channels when it is coexpressed in oocytes with either wild-type Kv2.2 or Kv2.1 subunits. These results indicate that Kv2.1 and Kv2.2 subunits are capable of heteromultimerization. Thus, in native cells either Kv2.1 and Kv2.2 subunits are targeted at an early stage to different biosynthetic compartments or heteromultimerization otherwise is inhibited.

Fig. 1.

Xenopus Kv2.1 and Kv2.2 transcripts induce the expression of functionally similar currents.A, Oocytes were injected with 0.2 ng of wild-type Kv2.1 RNA (left), 1.8 ng of wild-type Kv2.2 RNA (center), or 0.2 ng of Kv2.1 RNA plus 1.8 ng of Kv2.2 RNA (right). The injection of a mixture of wild-type Kv2.1 and Kv2.2 transcripts induces larger currents than either Kv2.1 or Kv2.2 RNA injected alone. Currents were generated in response to 160 msec voltage steps to potentials ranging from −50 to +100 mV from a holding potential of −80 mV; leak-subtracted currents are shown (see Materials and Methods). Calibration: 1.9 μA, 55 msec.B, The voltage-dependent properties of activation for the currents induced by the expression of wild-type Kv2.1 alone (n = 39) or a functionally equal amount of wild-type Kv2.1 and Kv2.2 RNA (n = 34) are shown on the left. Similar graphs for wild-type Kv2.2 currents (n = 31) and those induced by a functionally equal amount of wild-type Kv2.1 and Kv2.2 transcripts are shown on theright. Symbols are mean values; error bars indicate SD.

Fig. 2.

Amino acid substitutions in the pore region create nonfunctional Kv2.2 subunits. Currents are induced in the oocyte by the injection of 6.25 ng of wild-type Kv2.2 RNA (A) or 6.25 ng of each of the Kv2.2 pore mutants (C–F). Homomultimeric expression of WΔC Kv2.2, WΔF-YΔT Kv2.2, and WΔC-YΔT Kv2.2 transcripts does not result in the formation of functional channels (D–F), whereas injection of WΔF Kv2.2 RNA (C) induces currents with reduced amplitude as compared with wild-type Kv2.2. The injection of RNase-free water into the oocyte does not induce currents (B). Currents were generated in response to 60 msec voltage steps to potentials ranging from −50 to +100 mV from a holding potential of −80 mV; leak-subtracted currents are shown (see Materials and Methods). Calibration: 3.5 μA, 20 msec.

Fig. 3.

Double mutations in the Kv2.2 pore region are required for the construction of an efficient inhibitory subunit. Currents were recorded from oocytes injected with wild-type Kv2.2 transcripts alone (A) or wild-type Kv2.2 RNA mixed with each pore mutant in a 1:1 ratio (B–E). The amount of wild-type Kv2.2 RNA in each injection solution was kept constant. Coinjection of the WΔF Kv2.2 subunit caused no reduction in current amplitude as compared with wild-type Kv2.2 currents (B); coexpression of WΔC Kv2.2 and WΔF-YΔT Kv2.2 with wild-type Kv2.2 led to an intermediate reduction in current amplitude (C,D), and coinjection of WΔC-YΔT Kv2.2 transcripts caused the greatest decrease in current size (E). Currents were generated in response to 60 msec voltage steps to potentials ranging from −50 to +100 mV from a holding potential of −80 mV; leak-subtracted currents are shown (see Materials and Methods). Calibration: 3.5 μA, 20 msec.

Fig. 4.

WΔC-YΔT Kv2.2 behaves as expected for a dominant-negative subunit. Oocytes were injected with wild-type Kv2.2 RNA (A) or two different ratios of wild-type and WΔC-YΔT mutant transcripts (B, C). The amount of wild-type RNA in each injection solution was kept constant. Increasing the ratio of mutant to wild-type RNA led to the induction of smaller currents despite the increasing amount of total RNA injected into the oocyte. Currents were generated in response to 60 msec voltage steps to potentials ranging from −50 to +100 mV from a holding potential of −80 mV; leak-subtracted currents are shown (see Materials and Methods). Calibration: 2.7 μA, 20 msec.

Fig. 5.

Kv2.1 and Kv2.2 subunits coassemble. Oocytes were injected with wild-type Kv2.1 alone (A1), wild-type Kv2.2 alone (B1), a functionally equal amount of wild-type Kv2.1 and Kv2.2 RNA (A2), four times the functional amount of wild-type Kv2.2 as Kv2.1 RNA (A3), an equal amount of wild-type Kv2.2 and dominant-negative Kv2.2 RNA (B2), four times the amount of dominant-negative Kv2.2 RNA as wild-type Kv2.2 transcripts (B3), a functionally equal amount of wild-type Kv2.1 RNA and dominant-negative Kv2.2 RNA (C2), or four times the functionally equivalent amount of dominant-negative Kv2.2 transcripts as wild-type Kv2.2 RNA (C3). The dominant-negative Kv2.2 subunit causes a similar reduction in current amplitude when it is coexpressed with either wild-type Kv2.1 or wild-type Kv2.2 subunits (compareB2 and C2, B3 andC3) despite the increased amount of total RNA injected into the oocyte. Coexpression of wild-type Kv2.1 and Kv2.2 subunits leads to the induction of larger currents (A2,A3). In the diagram (C1), open circles represent wild-type Kv2.1 subunits, and filled circles represent dominant-negative Kv2.2 subunits. The heteromultimeric Kv2.1/dominant-negative Kv2.2 channels represented inbrackets indicate two possible arrangements for a heterotetramer containing two mutant subunits. Currents were generated in response to 160 msec voltage steps to potentials ranging from −50 to +100 mV from a holding potential of −80 mV; leak-subtracted currents are shown (see Materials and Methods). Calibration: 3.6 μA, 80 msec.

Fig. 6.

The conductance–voltage relations for wild-type Kv2.1 or wild-type Kv2.2 subunits expressed alone or in combination with WΔC-YΔT mutant or wild-type Kv2.1 and Kv2.2 subunits are similar. Conductance–voltage relations were plotted for wild-type Kv2.1 subunits alone or in combination with different ratios of dominant-negative mutant Kv2.2 subunits (A,B). Similar graphs were derived for wild-type Kv2.2 subunits injected into the oocyte alone or with different ratios of WΔC-YΔT Kv2.2 mutant subunits (C, D). The steady-state activation properties of the wild-type Kv2.1 or Kv2.2 current and the current remaining with the coexpression of either wild-type Kv2.1 or wild-type Kv2.2 and different ratios of the mutant Kv2.2 subunit are similar. This is the result expected if the subunit acts in a dominant-negative manner or does not demonstrate altered voltage-sensing properties. Symbols are mean values; error bars indicate SD. The SD bars are large for wild-type/WΔC-YΔT Kv2.2 mixtures (e.g., A, B). In these cases the current amplitudes are small, and the endogenous currents of the oocyte presumably contribute substantially to the overall current. The numbers of oocytes range from 17 to 39.