Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons

Abstract

We determined the expression of Kv2 channel subunits in rat somatosensory and motor cortex and tested for the contributions of Kv2 subunits to slowly inactivating K + currents in supragranular pyramidal neurons. Single cell RT-PCR showed that virtually all pyramidal cells expressed Kv2.1 mRNA and ∼80% expressed Kv2.2 mRNA. Immunocytochemistry revealed striking differences in the distribution of Kv2.1 and Kv2.2 subunits. Kv2.1 subunits were clustered and located on somata and proximal dendrites of all pyramidal cells. Kv2.2 subunits were primarily distributed on large apical dendrites of a subset of pyramidal cells from deep layers. We used two methods for isolating currents through Kv2 channels after excluding contributions from Kv1 subunits: intracellular diffusion of Kv2.1 antibodies through the recording pipette and extracellular application of rStromatoxin-1 (ScTx). The Kv2.1 antibody specifically blocked the slowly inactivating K + current by 25-50% (at 8 min), demonstrating that Kv2.1 subunits underlie much of this current in neocortical pyramidal neurons. ScTx (300 nM) also inhibited ∼40% of the slowly inactivating K + current. We observed occlusion between the actions of Kv2.1 antibody and ScTx. In addition, Kv2.1 antibody-and ScTx-sensitive currents demonstrated similar recovery from inactivation and voltage dependence and kinetics of activation and inactivation. These data indicate that both agents targeted the same channels. Considering the localization of Kv2.1 and 2.2 subunits, currents from truncated dissociated cells are probably dominated by Kv2.1 subunits. Compared with Kv2.1 currents in expression systems, the Kv2.1 current in neocortical pyramidal cells activated and inactivated at relatively negative potentials and was very sensitive to holding potential.

Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons

“…At this dose, ScTx also blocks A-type current in most cells (see below). The ScTx block is voltage dependent, with unblocking occurring at potentials more depolarized than approximately ϩ10 mV (Escoubas et al 2002;Guan et al 2007b).…”

“…ScTx inhibits the Kv2 subunit-mediated current in pyramidal neurons with an IC 50 of 160 nM (Guan et al 2007b). At 600 nM ScTx blocks Ͼ70% of the ScTx-sensitive current (Guan et al 2007b). ScTx doses higher than 600 nM would block more Kv2 channels but would increase experiment cost, and the subunit selectivity at high doses of ScTx is uncertain.…”

“…ScTx inhibits the Kv2 subunit-mediated current in pyramidal neurons with an IC 50 of 160 nM (Guan et al 2007b). At 600 nM ScTx blocks Ͼ70% of the ScTx-sensitive current (Guan et al 2007b).…”

“…BSA (0.1%) was added to the external solutions to prevent peptides from binding to glass and plastic vessels. We previously determined dose-response relationships for the blockers, and we chose doses that were specific for the relevant subunits (Guan et al 2006(Guan et al , 2007b. To study putative Kv1-mediated current (Guan et al 2006), we applied 100 nM ␣-dendrotoxin (DTX, blocks current through channels containing Kv1.1, Kv1.2, or Kv1.6 subunits; Harvey and Robertson 2004) and 10 -30 nM margatoxin (MTX, blocks Kv1.3-containing channels; Garcia-Calvo et al 1993).…”

“…Several voltage-gated potassium channel (Kv) subunits and currents have been identified in the pyramidal neurons of juvenile rat cortex (Bekkers 2000a(Bekkers , 2000bGuan et al 2005Guan et al , 2006Guan et al , 2007bHamill et al 1991;Korngreen and Sakmann 2000;Nerbonne et al 2008;Norris and Nerbonne 2010;Yuan et al 2005), and developmental changes in action potential (AP) and firing properties suggest that Kv channel subunits may be developmentally regulated. To help understand the functional roles of specific current components and to facilitate comparisons between studies reporting K ϩ currents from animals of different ages, we systematically characterized the expression of the total potassium current and the relative contributions of putative Kv1-and Kv2-mediated current and A-type current during the first 5 wk of postnatal development of pyramidal cells from rat somatosensory cortex.…”