Ih subunit characterisation in neontal rat dorsal root ganglion neurones

A. Momin1, H. Cadiou1, A. Mason2 and P.A. McNaughton1. 1Department of Pharmacology, Tennis Court Road, Cambridge, CB2 1PD, United Kingdom. 2Department of Molecular Pharmacology, Organon Laboratories Ltd, Newhouse, Lanarkshire, ML1 5SH, United Kingdom.

The hyperpolarisation-activated non-selective cation inward current (Ih) contributes to the resting membrane potential in sensory neurones. 4 genes responsible for Ih have been cloned and are termed hyperpolarisation-activated, cyclic nucleotide-gated (HCN1-4) channels. Each shows distinct kinetic properties and modulation by intracellular cAMP in expression systems. In the present study the whole-cell patch-clamp technique has been used to characterise native Ih from different size cell bodies of dorsal root ganglion (DRG) neurones. This work aimed to determine whether differences in Ih properties arise due to expression of distinctive isoforms of HCN.

DRG neurones were cultured from neonatal Wistar rats (3-7 days old and 10-25 grams) killed by cervical dislocation. DRG neurones were classified based upon cell body diameter as follows (µm): small (15-30), medium (30-37) and large (>37). Ih was activated by delivering 2 second hyperpolarisating potentials of -40 to -140mV in ­10mV increments from a holding potential of -60mV. All experiments performed at room temperature. Data are represented as mean ± S.E.M. Statistical analysis was performed using Student’s t-test.

Ih was present in 44, 87 and 100% of small, medium and large DRG neurones respectively. Following application of the adenylate cyclase activator forskolin (50 µM) there was no significant difference in half maximal voltage (V½) of Ih, (-89.8 ± 1.5 mV and -90.3 ± 1.8 mV) or in the fast activation time constant (τf ) measured at -120mV, (81.0 ± 12.5 ms and 76.3 ± 4.7 ms, P>0.05, n = 5) in large DRG neurones. In small DRG neurones forskolin caused a significant shift in V½ from -90.1 ± 0.8 mV to -74.0 ± 1.2 mV (P<0.001) and a decrease in τ from 529.2 ± 51.0 ms to 391.8 ± 29.9 ms (P<0.001, n = 7).

Two populations of medium DRG neurones existed. In one group forskolin failed to shift V½, (-91.9 ± 0.9 mV and -90.3 ± 1.8 mV, P>0.05, n = 5), while in another forskolin caused a positive shift in V½ (-90.1 ± 0.8 mV and -74.0 ± 1.2 mV) and a decrease in τf from 241.1 ± 26.8 ms to 169.1 ± 11.2 ms (P<0.001, n = 9).

Our results suggest a differential distribution of HCN proteins in small, medium and large DRG neurones. In large DRG neurones the HCN subunit has fast activation kinetics and is not modulated by an adenylate cyclase activator, similar to results obtained using HCN1 in expression systems (Chen et al., 2001). Small DRG neurones have relatively slower activation kinetics and are modulated by an adenylate cyclase activator, a feature seen with HCN2 and 4 expression. Medium DRG neurones fall into either one of these categories.

Chen, S. et al. (2001). J. Gen. Physiol. 117, 491-503.

Work was supported by the B.B.S.R.C and Organon Laboratories Limited.