Single-channel properties of native and cloned rat vanilloid receptors

Abstract

The responses of single-channel currents to capsaicin were recorded using the giga-seal patch-clamp technique in cell-attached and excised (inside-out/outside-out) patches from embryonic rat dorsal root ganglion (DRG) neurones in culture and in Xenopus oocytes heterologously expressing the rat vanilloid receptor (rVR1). Native and cloned vanilloid receptor (VR)-mediated currents exhibited outward rectification. In both the DRG neurones and oocytes expressing VR1, the chord conductances at -60 and +60 mV were approximately 50 and approximately 100 pS, respectively. At positive potentials, the channel exhibited a single conductance state. In contrast, at negative potentials, brief sojourns to subconductance states were apparent. The probability of the channel being open (P(o)) was dependent on the transmembrane voltage and the patch configuration (i.e. cell-attached vs. excised). In both DRG neurones and oocytes, the P(o) was greater at positive (+60 mV) than at negative (-60 mV) potentials. In cell-attached patches, the P(o) was approximately twofold higher, regardless of the applied potential. Most likely, the outward rectification observed in whole-cell currents is due to the voltage dependence of single-channel conductance and P(o). The open-time distributions of single-channel currents recorded from native and cloned VRs in the presence of low agonist concentrations (0.01-0.03 microM) were best fitted with three exponential components. The closed-time distributions were best fitted by five exponential components. At higher concentrations (0.5-1 microM), an additional component was required to fit the open-time distribution, and the number of exponential components needed to fit the closed-time distributions was reduced to two. The overall mean open time at +60 mV was approximately 4 ms, compared to approximately 1.2 ms at -60 mV. However, the overall mean closed time was not voltage dependent. There were no significant differences between the native and cloned receptors. A comparison of single-channel properties of native and heterologously expressed VR channels indicates that expression of the rVR1 subunit alone can account for the single-channel behaviour of the majority of the native VRs. These results suggest that either native VRs are made up of VR1 subunits, or the incorporation of subunits other than VR1 does not influence the functional properties. The responses of single-channel currents to capsaicin were recorded using the giga-seal patch-clamp technique in cell-attached and excised (inside-out/outside-out) patches from embryonic rat dorsal root ganglion (DRG) neurones in culture and in Xenopus oocytes heterologously expressing the rat vanilloid receptor (rVR1). Native and cloned vanilloid receptor (VR)-mediated currents exhibited outward rectification. In both the DRG neurones and oocytes expressing VR1, the chord conductances at -60 and +60 mV were approximately 50 and approximately 100 pS, respectively. At positive potentials, the channel exhibited a single conductance state. In contrast, at negative potentials, brief sojourns to subconductance states were apparent. The probability of the channel being open (P(o)) was dependent on the transmembrane voltage and the patch configuration (i.e. cell-attached vs. excised). In both DRG neurones and oocytes, the P(o) was greater at positive (+60 mV) than at negative (-60 mV) potentials. In cell-attached patches, the P(o) was approximately twofold higher, regardless of the applied potential. Most likely, the outward rectification observed in whole-cell currents is due to the voltage dependence of single-channel conductance and P(o). The open-time distributions of single-channel currents recorded from native and cloned VRs in the presence of low agonist concentrations (0.01-0.03 microM) were best fitted with three exponential components. The closed-time distributions were best fitted by five exponential components. At higher concentrations (0.5-1 microM), an additional component was required to fit the open-time distribution, and the number of exponential components needed to fit the closed-time distributions was reduced to two. The overall mean open time at +60 mV was approximately 4 ms, compared to approximately 1.2 ms at -60 mV. However, the overall mean closed time was not voltage dependent. There were no significant differences between the native and cloned receptors. A comparison of single-channel properties of native and heterologously expressed VR channels indicates that expression of the rVR1 subunit alone can account for the single-channel behaviour of the majority of the native VRs. These results suggest that either native VRs are made up of VR1 subunits, or the incorporation of subunits other than VR1 does not influence the functional properties.

Figure 1. Single-channel currents activated by capsaicin

Application of capsaicin (1 μm) to an outside-out patch obtained from a Xenopus oocyte injected with vanilloid receptor (VR)1 cRNA activated single-channel currents. A, at −80 mV the current amplitude is 4.5 pA, corresponding to a conductance of 86 pS. B, at +80 mV the current amplitude was 8.5 mV, corresponding to single-channel conductance of 106 pS.

Figure 2. Subconductance levels of VR1 channel

Single-channel currents recorded at −80 mV show sojourns to multiple conductance levels. Selected openings that show clear sublevels are grouped and the amplitude histogram (inset) shows two additional conductance levels (5.3, 3.6 and 1.7 pA corresponding to 66, 45 and 21 pS, respectively). * Regions that have been shown below in an expanded time scale.

Figure 3. The single-channel probability of opening (P_o) is dependent on the patch configuration and transmembrane voltage

A, single-channel current recordings at +60 and −60 mV from dorsal root ganglion (DRG) neurones with the pipette containing 500 nm capsaicin. B, current recordings after forming an inside-out patch. C, in oocytes heterologously expressing VR1, single-channel currents activated by 500 nm capsaicin at +60 and −60 mV. D, current recordings after forming an inside-out patch. In cell-attached patches the P_o was higher than in excised patches, and at positive potentials the P_o was higher than at negative potentials.

Figure 4. Single-channel P_o in cell-attached patches and after patch excision

In the presence of 500 nm capsaicin, the P_o was higher in cell-attached patches (> 0.7) and remained higher (▪), but upon patch excision, the P_o decreased over time (○). Time zero represents the beginning time for both cell-attached as well as excised patches.

Figure 5. Outward rectification of the VR in DRG neurones and in oocytes heterologously expressing VR1

A, single-channel current recorded at different membrane potentials from DRG neurones. Both single-channel P_o (B) and conductance (C) were reduced at hyperpolarised potentials. D, single-channel current recorded at different membrane potentials from an oocyte heterologously expressing VR1. E, progressive reduction in P_o with hyperpolarisation. F, current-voltage relationship showing that the single-channel conductance was smaller at negative potentials. It is clear that both single-channel conductance and P_o contributed to the macroscopic current rectification.

Figure 6. Kinetic analysis of single-channel currents activated by capsaicin from native VRs in DRG neurones and cloned VR1 heterologously expressed in oocytes

A, in DRG neurones, at positive potentials (+60 mV) the open- and closed-time distributions are well fitted with three and five exponential components, respectively. B, at negative potentials (-60 mV) the open- and closed-time distributions are well fitted with three and four exponential components, respectively. C, in oocytes expressing VR1, at positive potentials (+60 mV) open- and closed-time distributions are well fitted with four and five exponential components, respectively. D, at negative potentials (-60 mV), the open- and closed-time distributions are well fitted with three and four exponential components, respectively. The time constants of the exponential components and the relative fraction (in parenthesis) are shown.