Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in neurons
Figure Lengend Snippet: Cav1.2 channel activity is increased by coexpression with Kv2.1 P404W . (A) Representative Ca 2+ current trace families recorded from HEK293T cells transfected with Cav1.2-GFP and auxiliary subunits Cavβ3 and Cavα 2 δ 1 , without (+ pcDNA3 empty vector) with cotransfection of DsRed-Kv2.1 P404W . For panels D-F, data are from cells without (+ pcDNA3 empty vector, in black) or with coexpression of Kv2.1 P404W (in red). (B) Normalized current-voltage ( I-V ) relationship of whole-cell I Ca recorded from n=17 (Cav1.2 + pcDNA3) and n =10 (Cav1.2 + Kv2.1 P404W ) cells. (C) Voltage-dependence of whole cell Cav1.2 conductance G / G max and steady-state inactivation I / I max . For the conductance-voltage relationships, the half-maximal activation voltage V 1/2 =-8.9±0.8 [pcDNA3] vs. -13.9±1.6 [+Kv2.1 P404W ] mV, p=0.0045; slope factor k =6.9±0.3 [pcDNA3] vs. 4.5±0.7 [+Kv2.1 P404W ], p=0.0025; Student’s t-test. (D) Representative nitrendipine-sensitive Cav1.2 gating and tail currents recorded from control (pcDNA3) cells and cells coexpressing Kv2.1 P404W . (E) Quantification of nitrendipine-sensitive Cav1.2 Q on (left), I tail (center), and Q on vs. I tail (right).. Each point corresponds to a single cell (*p=0.019, Student’s t -test). (F) Average Rhod-2 fluorescence intensity measurements obtained from cells held at different membrane potentials during voltage clamp experiments ( n =4 cells per condition). (G) Average fluorescence intensity measurements from Fluo4-loaded HEK293T cells transfected with Cav1.2, auxiliary subunits Cavβ3 and Cavα2δ, without (+ pcDNA3 empty vector, in black) or with Cotransfection of Kv2.1 WT (in blue) or Kv2.1 P404W (in red). Ca 2+ influx was stimulated by depolarization with high extracellular K + (45 mM) as indicated on the graph. (H) Average peak fluorescence values obtained during high-K + depolarization of HEK293T cells expressing Cav1.2 and Kv2.1 WT or Kv2.1 P404W as in G. Each point corresponds to a single cell. Bars are mean ± SD (**p
Article Snippet: To isolate gating currents and I tail produced by Cav1.2, we subtracted currents measured in the presence of nitrendipine from those measured in KRB alone.
Techniques: Activity Assay, Transfection, Plasmid Preparation, Cotransfection, Activation Assay, Fluorescence, Expressing