Fig 1.
Ics in cortical layer 5 pyramidal neurons.
A: Whole cell, voltage clamp recording while blocking Na+ currents with TTX (1μM), Ca2+ currents with Cd2+ (200μM) and Cs+ (135mM) in the recording pipette to block K+ currents. Voltage steps (10 mV increments) revealed an outward current at voltages more depolarized than -20mV. B: In the same cell, a slow (110 mV/s) voltage ramp (-70 to +40 mV) generated an outward rectifying instantaneous IV curve similar to the one in A (red squares). C: Repolarizing voltage steps from +40 mV (10 mV increments) reveal tail currents at potentials more negative than -40 mV (left). Right: IV curve for this cell shows reversal at around -40 mV. Inset: distribution of reversal potentials of Ics for 8 neurons.
Fig 2.
Voltage-dependent activation and deactivation of Ics.
A: Response of a representative neuron to depolarizing voltage steps from a holding potential of -70 mV. Application of voltage steps (lower panel) resulted in outward currents (upper panel, solid lines) which were fitted mono-exponentially (dashed lines). B: Hyperpolarizing steps (lower panel) from +20 mV, resulted in inward tail currents (upper panel, solid lines). Deactivation of Ics was voltage-dependent and was also fitted with single exponentials (dashed lines). C: Time constants of activation (empty squares) and deactivation (filled squares) as a function of membrane voltage (n = 5 neurons).
Fig 3.
Ics is not associated with detectible influx of Ca2+ or Na+.
A: Image: A layer 5 neuron during fluorescent Ca2+ imaging with OGB-1 in the recording electrode. Traces: -70 to +40 mV ramps resulted in Ca2+ influx (upper black trace) and electrically recorded Ca2+ action currents (middle black trace). Adding 200μM Cd2+ to the bath completely eliminated the Ca2+ flux (upper red trace), leaving only Ics (middle red trace). B: Image: A layer 5 neuron during fluorescent Na+ imaging with SBFI in the recording electrode. Traces: -70 to 0 mV ramps resulted in Na+ influx and (upper black trace) and electrically recorded persistent Na+ current (middle black trace). Adding 1μM TTX completely eliminated both the Na+ flux and the persistent Na+ current.
Fig 4.
Pharmacological sensitivity of Ics.
A: Changes (as compared to break-in) in current amplitude at +40 mV in response to the potential Ics antagonists: carbenoxelone (100 μM), SKF96365 (100 μM), 2-APB (100 μM), La3+ (1 mM) and TEA (40 mM). La3+ concentration was reduced to 100 μM when co-applied with TEA. B: Comparison of current amplitudes at +40 mV when recording electrodes contained either Cs+ (135 mM) or Cs+ + TEA (30 mM of TEA replaced) to block K+ channels. P values represent comparison to control, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001). Data shown are averages and error bars represent s.e.m.
Fig 5.
Layer 5 neurons I-V curves are similar in neurons lacking TRPC1 or TRPC5 channels.
Left: representative instantaneous I-V curves during slow ramps from wild type (black), TRPC1 KO (red) or TRPC5 KO (blue). Right: IV curve shape for the groups, as quantified by the rectification index (see results) is not significantly different. Data shown are averages and error bars represent s.e.m. (P > 0.05).