Identification of a molecular gating determinant within the carboxy terminal region of Cav3.3 T-type channels

Figures

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  Fig. 1. Identification of a conserved region within the carboxyl terminal domain of T-type channels. a Schematic representation of the T-type channel membrane topology and protein sequence alignment of the carboxyl terminal domain of the three T-type channel isoforms (Cav3.1, Cav3.2, and Cav3.3) showing a stretch of highly conserved residues within the proximal region (highlighted in yellow). b Protein sequence alignment of the conserved proximal C-terminal region of Cav3.3 with the C-terminal domain of high-voltage-activated (HVA) calcium channels. Only HVA channels for which some degree of homology exist are shown. Numbers in bracket indicate the percentage of identity and similarity, respectively

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  Fig. 2. Role of C-terminus on Cav3.3 activation. a Representative set of current traces recorded in response to 300 ms depolarizing steps ranging from − 80 mV to 0 mV from a holding potential of − 100 mV from cells expressing the different Cav3.3 channel combinations indicated above the current traces. The red trace indicates the current recorded in response to a depolarization step at − 50 mV. b Corresponding mean normalized voltage dependence of activation. c Corresponding mean half activation potential. d Corresponding mean activation slope factor. e Current-voltage relationships for cells expressing WT (black circles), C short (grey circles), and ΔC (purple circles) channel constructs. f Corresponding mean maximal conductance

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  Fig. 3. Role of C-terminus on Cav3.3 inactivation a Representative set of current traces recorded in response to 300 ms depolarizing step to − 20 mV from a holding potential of − 100 mV, preceded by 15 s inactivating pulse ranging from − 120 mV to − 30 mV from cells expressing the different Cav3.3 channel combinations indicated above the current traces. The red trace indicates the current recorded after an inactivating pulse at − 60 mV. b Corresponding mean normalized voltage dependence of steady state inactivation. c Corresponding mean half inactivation potential. d Corresponding mean steady state inactivation slope factor

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  Fig. 4. Role of C-terminus on Cav3.3 inactivation kinetics. a Representative current traces recorded in response to 1 s depolarizing step to − 20 mV from a holding potential of − 100 mV from cells expressing the different Cav3.3 channel combinations indicated above. The dotted red line indicates the fit of the current decay by a single (WT, C short, and ΔC + CD4-C) or double exponential function (ΔC). The grey line represents the WT current for comparison. b Corresponding mean time constant of inactivation. c Corresponding mean fractions of inactivating and non-inactivating current

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  Fig. 5. Role of C-terminus on Cav3.3 recovery from inactivation. a Representative current traces recorded in response to a 300 ms depolarizing step to − 20 mV preceded by a recovery pulse at − 110 mV for a duration ranging between 0.1 ms and 2500 ms after a 15 s period of inactivation at 0 mV from cells expressing the different Cav3.3 channel combinations indicated above the current traces. The red line represents the current recorded after 200 ms recovery. b Corresponding mean normalized recovery from inactivation. c Corresponding mean time constant of recovery from inactivation

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  Fig. 6. Role of C-terminus on Cav3.3 window current. a Window current (red) obtained from the mean activation and inactivation curves for Cav3.3 channel combination indicated above. b Amplitude of the window current measured as the area of the overlap between the activation and inactivation curves. c Mid voltage of the window current measured at the intersection between the activation and inactivation curves

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  Fig. 7. Role of C-terminus on Cav3.3 gating currents. a Representative gating current traces recorded from cells expressing the various Cav3.3 variants. Each trace represents an average of five traces recorded during cell depolarization to its reversal potential. Below each current trace is a depiction of time courses of integrals calculated for that trace. Current traces and integrals are scaled to 1 for a better comparison. Grey lines represent the integral of the gating current trace for the WT channel. b Corresponding mean Gmax/Qrev ratios calculated for each investigated cell. c Corresponding mean 10–90% rise times calculated from the integral time course shown in panel a