Molecular Heterogeneity of the Voltage-Gated Fast Transient Outward K⁺ Current, I_Af, in Mammalian Neurons

Distinct components ofI_Af inactivation in SCG neurons. The time constants of inactivation of the rapid component of current decay,I_Af, were determined in wild-type, Kv4.2W362F-expressing, and Kv1.5W461F-expressing SCG cells as described in Materials and Methods. The τ_decay values were binned in 40 msec increments for comparison purposes and, as is evident, the distributions of τ_decay values in wild-type, Kv4.2W362F-expressing, and Kv1.5W461F-expressing SCG cells are distinct.

Expression of Kv1 α-subunits in SCG neurons. Isolated wild-type SCG neurons were fixed and probed with polyclonal antibodies generated against Kv1.2 (A), Kv1.4 (B), or Kv1.5 (C) 48 hr after plating, as described in Materials and Methods. As is evident, the Kv1.2 and Kv1.4 α-subunits are readily detected in SCG neurons, although the staining patterns of these subunits are distinct. Scale bars, 50 μm.

Expression of Kv1.5W461F-myc in transfected SCG neurons. Isolated SCG neurons, transfected with EGFP alone (top) or with Kv1.5W461F-myc and EGFP (bottom) using the gene gun, were fixed and stained 24 hr later (see Materials and Methods). The top andbottom panels show EGFP fluorescence (left) and Cy3 fluorescence (right) images of the same field. Anti-myc staining is only evident in cultures transfected with Kv1.5W461F-myc (compare right panels,top and bottom). In addition, EGFP expression correlates with Kv1.5W461F (bottom, compare left and right panels). Scale bar, 50 μm.

Coexpression of both Kv1.5W461F and Kv4.2W362F eliminates I_Af. Isolated SCG neurons were transfected with Kv1.5W461F, Kv4.2W362F, and EGFP using the Biolistic Gene Gun (as described in Materials and Methods), and outward K⁺ currents were recorded from EGFP-positive cells as described in the legend to Figure 1. Two distinct current waveforms were evident in these recordings: the vast majority (76%) of cells were found to express only I_K andI_SS and were classified as type III (B); the remaining cells (24%) expressI_As,I_K, andI_SS, and, therefore, are type II cells lacking I_Af (A). The fast transient current I_Af was not detected in any of these cells (n = 15). Analysis of the decay phases of the currents revealed that the densities ofI_As,I_K, andI_SS in Kv1.5W461F + Kv4.2W362F-expressing type II and III cells are indistinguishable from the currents in wild-type II and III cells (Table 1).

Expression of Kv1.5W461F increases the percentage of adapting cells. Isolated SCG neurons were transfected with Kv1.5W461F (and EGFP), and action potentials and repetitive firing patterns were recorded in response to brief or prolonged depolarizing current injections, as described in Materials and Methods. Current-clamp recordings from (3) representative Kv1.5W461F-expressing cells are shown in A, B, andC. In each cell, single action potentials were elicited by 1.5 msec current injections (left), and repetitive firing patterns were recorded in response to 100 pA (middle) or 200 pA (right) 500 msec current injections. On the basis of the response(s) to the 500 msec current injections, cells were classified as phasic (A), adapting (B), or tonic (C) (Table 2). With Kv1.5W461F expression, the percentages of adapting and tonic cells are increased, and the percentage of phasic cells is decreased relative to the firing pattern distribution seen in wild-type cells (Table 2).

Elimination of the Kv1- and Kv4-encodedI_Af increases the percentage of adapting SCG neurons. Action potentials and repetitive firing patterns were recorded from isolated SCG neurons 24 hr after transfection with EGFP alone (A), EGFP and Kv4.2W362F (B), EGFP and Kv1.5W461F (C), or EGFP, Kv1.5W461F and Kv4.2W362F (D), as described in the legend to Figure 6. The phasic (left), adapting (middle), and tonic (right) firing patterns were evident in recordings obtained under all of these experimental conditions. In recordings from cells with reduced I_Af density, the percentages of phasic cells are lower, and the percentages of adapting and tonic cells are higher than seen in recordings from wild-type cells (Tables 2, 4, 5).

Expression of Kv1.4 increases the percentage of phasic firing SCG cells. Action potentials and repetitive firing patterns, recorded as described in the legend to Figure 6, were obtained from isolated SCG neurons 24 hr after transfection with wild-type Kv1.4 and EGFP. As in wild-type cells, the phasic (A), adapting (B), and tonic (C) firing patterns were seen in recordings from cells transfected with Kv1.4. The percentage of adapting cells, however, is lower and the percentage of phasic cells is higher in Kv1.4-expressing cells than seen in recordings from wild-type or Kv1.5W461F-expressing SCG cells (Figs. 6, 7, Tables 2, 4).