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. 2005 Oct 1;568(Pt 1):155-69.
doi: 10.1113/jphysiol.2005.090951. Epub 2005 Jul 14.

Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes

Sohag Saleh  1 , Shuk Yin M Yeung, Sally Prestwich, Vladimír Pucovsky, Iain Greenwood
Affiliations

Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes

Sohag Saleh et al. J Physiol. .

Abstract

A voltage-gated Na+ current was characterised in freshly dissociated mouse portal vein (PV) smooth muscle myocytes. The current was found superimposed upon the relatively slow L-type Ca2+ current and was resistant to conventional Ca2+ channel blockers but was abolished by external Na+ replacement and tetrodotoxin (TTX, 1 microM). The molecular identity of the channel responsible for this conductance was determined by RT-PCR where only the transcripts for Na+ channel genes SCN7a, 8a and 9a were detected. The presence of the protein counterparts to the SCN8a and 9a genes (NaV1.6 and NaV1.7, respectively) on the individual smooth muscle myocytes were confirmed in immunocytochemistry, which showed diffuse staining around a predominantly plasmalemmal location. TTX inhibited the action potential in individual myocytes generated in the current clamp mode but isometric tissue tension experiments revealed that TTX (1 and 5 microM) had no effect on the inherent mouse PV rhythmicity. However, the Na+ channel opener veratridine (10 and 50 microM) significantly increased the length of contraction and the interval between contractions. This effect was not influenced by pre-incubation with atropine, prazosin and propranolol, but was reversed by TTX (1 microM) and completely abolished by nicardipine (1 microM). Furthermore, preincubation with the reverse-mode Na+-Ca2+ exchange blocker KB-R7943 (10 microM) also inhibited the veratridine response. We have established for the first time the molecular identity of the voltage-gated Na+ channel in freshly dispersed smooth muscle cells and have shown that these channels can modulate contractility through a novel mechanism of action possibly involving reverse mode Na+-Ca2+ exchange.

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Figures

Figure 1
Figure 1. Identification of a ‘fast’ nicardipine-resistant voltage-dependent current in freshly dissociated murine portal vein smooth muscle myocytes
A, a representative trace of an L-type Ca2+ current (ICaL), which was seen in 74% of cells in the voltage clamp mode using the perforated patch configuration. The protocol involved a step from −60 mV to +20 mV and the inward current was abolished by nicardipine (1 μm). B, example traces of the currents seen in 16% of cells under identical conditions and using the same protocol described for A. The relatively slow ICaL was superimposed on another component that had faster kinetics (IFAST). IFAST was not dihydropyridine-sensitive (1 μm nicardipine) and was not inhibited by 1 mm CdCl2 (C). The unlabelled scalars represent 50 pA and 250 ms. D, the distribution of current types seen in cells accessed in the perforated patch configuration and voltage clamped at a holding potential of −60 mV. Those with properties similar to A were categorised as L-type Ca2+ only, whilst waveforms similar to B and C were considered biphasic. A small percentage of cells also showed no inward current upon depolarization (total n = 192).
Figure 2
Figure 2. Identification of IFAST
A, the effect of external Na+ ion replacement by TRIS on biphasic waveforms generated by depolarization from −60 to 20 mV. In a the effect is shown on both components of the biphasic waveform whereas b shows the effect on a current where the slower ICaL component had been previously removed by application of 1 μm nicardipine (nic). In both incidences the fast component of current was eliminated when the external Na+ was removed. Ba, representative trace from a cell containing IFAST where ICaL has been eliminated by preapplication with nicardipine and the effect of increasing concentrations of tetrodotoxin (TTX) on IFAST amplitude are apparent. b, log concentration–effect plot for TTX with the data points fitted with a Hill coefficient and n values labelled at each point. The IC50 for TTX inhibition was calculated to be 12 ± 1 nm.
Figure 3
Figure 3. Biophysical properties of IFAST
A, the current–voltage (I–V) relationship of IFAST measured at the peak inward current in the presence of 1 μm nicardipine (•) and the corresponding cells in a Na+ free external solution (▵). Removal of Na+ from the external solution abolished IFAST leaving a small outward current at negative potentials. B, a bar graph illustrating the mean voltage dependent deactivation kinetics of IFAST measured at the voltage steps indicated from a Vh=−60 mV (n = 5) calculated by fitting the data with a single exponential. C, the activation and inactivation of the channel in the presence of 1 μm nicardipine. The mean peak current values were established using a double pulse protocol in which cells were stepped from −60 mV to a range of voltages between −100 and +40 mV for 1.5 s, followed by a 250 ms step to +20 mV. Inactivation values (○) and activation (▪) were normalised to the maximal evoked current and the mean data fitted with a Boltzmann function. All data are represented as means ±s.e.m.
Figure 4
Figure 4. Molecular identification of voltage- dependent Na+ channel subunits expressed in murine portal vein
Reverse transcriptase polymerase chain reaction was undertaken using primers specific for SCNa isoforms and mRNA extracted from freshly dissected mouse heart, brain and portal vein. The bands have been visualised on a UV transilluminator using ethidium bromide incorporated into agarose gels. Positive signals for each Na+ channel isoform detected are included in the accompanying text.
Figure 5
Figure 5. Immunocytochemical staining of murine portal vein smooth muscle cells for NaV1.6 and NaV1.7 type of voltage-dependent sodium channels
The fluorescence images of a single confocal plane of the cell are labelled with NaV1.6 antibodies (A); NaV1.6 antibodies preincubated with their antigenic peptide (B); NaV1.7 antibodies (C); and NaV1.7 antibodies preincubated with their antigenic peptide (D). Circles in A indicate Regions 1 and 2 that were used to analyse the localization of fluorescence (for details see Methods). The calibration in A–D is 10 μm. A dotted line has been used in B and D to outline the contour of a cell, due to its low fluorescence. E, the summarized data on localization of NaV1.6 and NaV1.7 fluorescence in the cells. There was significantly more fluorescence in the region within ∼1.6 μm of plasma membrane (Region 1) than in the deep cytoplasm (Region 2) or when compared with whole cell average. F, summary data on intensity of fluorescence, expressed as average pixel fluorescence (intensity units per pixel). The values of all pixels in the cell's confocal plane were added up and then divided by the number of such pixels. The specificity of labelling was confirmed for both antibodies by greatly reduced fluorescence after preincubation with respective antigenic peptide or by virtual lack of fluorescence in the absence of primary antibodies. *Statistical significance.
Figure 6
Figure 6. Effect of TTX on action potential generation and portal vein rhythmicity
Aa, representative family of voltage responses generated by applying 2 ms pulses of current in 25 pA increments (see inset) on a cell held in the current clamp mode. The bold trace is indicative of a control (as labelled) action potential (AP) generated by applying 75 pA for 2 ms. Membrane potentials elicited by this protocol in the presence of 1 μm TTX are shown in the right-hand panel. The highlighted trace is the voltage response evoked by 2 ms injection of 75 pA. Ab, another family of currents generated under the same conditions using the protocol previously described. Upon application of 2 μm nicardipine (right-hand panel) it was no longer possible to generate an AP even with a 125 pA injection of current. This effect was consistently reproducible (n = 4). B, a representative isometric tension recording from whole murine portal vein where each vertical deflection represents a spontaneous increase in tension. TTX at 1 and 5 μm was applied for the period denoted by the horizontal bars and had no significant effect on the spontaneous contractions of the portal vein. Unlabelled scalars represent 75 mg and 2 min.
Figure 7
Figure 7. Analysis of the effect of veratridine on whole portal veins
A, representative trace from an isometric tension recording showing the effect of veratridine, which significantly altered the contraction frequency, the duration of contraction and the interval between contractions in all tissues (see text, n = 6). These effects were reversed by the application of 1 μm TTX (n = 5). The inset shows a magnification of the sections indicated. The scalars represent 50 mg and 2 min for the long-term trace and 100 mg and 30 s for the inset. B, trace showing the effects of applying 5 μm atropine (At), 5 μm propranolol (Pl) and 5 μm prazosin (Pz) to the PV. The effect of veratridine (Vt) was not affected by preincubation with these reagents demonstrating that it was independent from cholinergic or adrenergic influence. Similar effects were observed in 2 other tissues. The scalars represent 30 mg and 3 min. C, the effect of 50 μm veratridine on spontaneous contractions under the same conditions as the previous traces. In this example application of 1 μm nicardipine not only reversed the veratridine product but also resulted in an almost complete abolition of all contractions.
Figure 8
Figure 8. The effects of K- BR7943
A, bar charts representing mean data (±s.e.m., n = 8) showing the effect of veratridine on the interval between contractions (a) and the duration of contractions (b) under normal conditions and following 10 min of preincubation with the reverse mode Na+–Ca2+ exchange blocker KB-R7843 (10 μm as labelled). B, the effect of 10 μm KB-R7943 (KBR) on IFAST evoked by depolarization from −90 mV (as labelled). a, traces showing the decrease in IFAST amplitude upon application of KBR and the subsequent increase upon washout. b, an example of a cell where KBR did not inhibit IFAST amplitude but this agent increased the apparent decay of the evoked current.
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