Localization-dependent activity of the Kv2.1 delayed-rectifier K+ channel

Abstract

The Kv2.1 K(+) channel is highly expressed throughout the brain, where it regulates excitability during periods of high-frequency stimulation. Kv2.1 is unique among Kv channels in that it targets to large surface clusters on the neuronal soma and proximal dendrites. These clusters also form in transfected HEK cells. Following excessive excitatory stimulation, Kv2.1 declusters with an accompanying 20- to 30-mV hyperpolarizing shift in the activation threshold. Although most Kv2.1 channels are clustered, there is a pool of Kv2.1 resident outside of these domains. Using the cell-attached patch clamp technique, we investigated the hypothesis that Kv2.1 activity varies as a function of cell surface location. We found that clustered Kv2.1 channels do not efficiently conduct K(+), whereas the nonclustered channels are responsible for the high threshold delayed rectifier K(+) current typical of Kv2.1. Comparison of gating and ionic currents indicates only 2% of the surface channels conduct, suggesting that the clustered channels still respond to membrane potential changes. Declustering induced via either actin depolymerization or alkaline phosphatase treatment did not increase whole-cell currents. Dephosphorylation resulted in a 25-mV hyperpolarizing shift, whereas actin depolymerization did not alter the activation midpoint. Taken together, these data demonstrate that clusters do not contain high threshold Kv2.1 channels whose voltage sensitivity shifts upon declustering; nor are they a reservoir of nonconducting channels that are activated upon release. On the basis of these findings, we propose unique roles for the clustered Kv2.1 that are independent of K(+) conductance.

Fig. 1.

Kv2.1 channels retained within cell surface clusters are nonconducting. (A) Overlay of the GFP fluorescence (green) and DIC images of the apical surface of a GFP-Kv2.1-loopBAD-expressing HEK cell during an on-cluster cell-attached patch-clamp experiment. The white spots are due to diffracted light from the patch pipette. (B) Fluorescence only image of the GFP-Kv2.1-loopBAD cluster targeted for cell-attached patch clamp. The red circle indicates the position of the patch pipette. (C) Representative sweeps at −15 mV from the cell in A. Upward deflections indicate channel opening, dashed line is zero current. (D) Ensemble average of 71 sweeps containing Kv2.1 channel activity. (E) Current–voltage relationship for all cell-attached on-cluster patches, n = 32. Data for each point are derived from a Gaussian fit to an all-points histogram of the total dataset at each potential. Error bars are smaller than the symbols. The slope conductance is 7.1 pS. (F) Ramp depolarization of an on-cluster patch from −75 to +125 mV at 0.05 mV/ms. Channel activity (upward deflection) is observed between −30 and +5 mV and again at +120 mV.

Fig. 2.

Nonclustered Kv2.1 channels are conducting K⁺ channels. (A) DIC and GFP fluorescence images of the apical surface of a GFP-Kv2.1-loopBAD-expressing HEK cell. The red circle in the GFP fluorescence image shows the location of the patch pipette, demonstrating there are no Kv2.1 clusters near the patch pipette. (The scale bar applies to both micrographs.) (B) Representative sweeps to the indicated potentials from an off-cluster patch. (C) Current–voltage relationship from four off-cluster patches. (D) Conductance–voltage relationship calculated from the data in C, assuming an intracellular [K⁺] of 140 mM and thus a reversal potential of −84 mV. V_1/2 of the fitted data from conductance–voltage plot = +10.6 mV. The red plot illustrates the voltage dependence as obtained from whole-cell currents illustrated in Fig. S2.

Fig. 3.

Nonconducting Kv2.1 channels on the cell surface respond to changes in membrane potential. (A) Representative gating currents at the indicated potentials for a GFP-Kv2.1-loopBAD-expressing HEK cell. (Scale bars: 1 nA, 50 ms.) (Inset) “On” gating current at +60 mV. (Scale bars: 1 pA, 2 ms.) (B) Normalized charge–voltage (Q–V) curve for GFP-Kv2.1-loopBAD. Q_max = 2.269 ± 571 nC, V_1/2 = −27.0 ± 4.0 mV (n = 8, P < 0.01, compared with V_G1/2). A fit to a single Boltzmann is shown because a fit of two distributions was not better. Red line is the fit from the conductance–voltage plot in Fig. S2B. (C) Scatter plot of the estimate of channel number from gating currents or ionic currents. The red line is the mean for each group. A value of 12.5 q_e per Kv2.1 channel was used to estimate channel number. P < 0.01.

Fig. 4.

Kv2.1 declustering does not increase whole-cell current. GFP-Kv2.1-loopBAD-expressing HEK cells were imaged for GFP before and during whole-cell voltage clamp. (A) Cell with prominent surface clusters before whole-cell voltage clamp and actin depolymerization. (B) The same cell 25 min after the addition of 200 nM swinholide A. (C) Outward currents observed at +60 mV before and after swinholide A-induced declustering. (D) Cell with prominent surface clusters before alkaline phosphatase dialysis (500 units/mL in the intracellular pipette solution) and whole-cell voltage clamp. (E) The same cell 15 min after membrane rupture. (F) Outward currents observed at +60 mV before (1 min after break-in) and after phosphatase-induced declustering (after 15 min of whole-cell dialysis).