Two isoforms of the hNK1R differing only in the length of C-terminal domain have been described (Fong et al., 1991). Recently, the mRNA expression pattern of the short isoform has been reported in discrete regions of the human brain (Caberlotto et al., 2003). However, the functional role of the short NK1R has not yet been elucidated. The aim of the present study was to investigate the protein expression and pharmacological properties of the short hNK1R in comparison with the long isoform after transient transfections in CHO-K1 and COS-7 cells.

Cells were transfected with pcDNA3.0 encoding the long or pcDNA 3.1 encoding the short hNK1R using FuGENE™6. Assays were carried out 48h after transfection. Gene transcription was confirmed by RT-PCR using primers spanning a sequence common to both isoforms (forward 5’- ccaaacatctccactaacacctc-3’, reverse 5’- tagaacaggccgtagtaccattc-3’). In binding experiments, cells were incubated for 1h at 25ºC with 30nM [³H]-Substance P (SP) and filtered through GF/C filters. The [³H]-SP concentration was 4 fold greater than that used by Fong et al. (1991) for the binding detection of the short and 30 fold the K_d of the long isoform. In functional experiments, the increase in [Ca²⁺]_i was detected with FLIPR (Molecular Devices). Cells were labelled with 2µM Fluo-4 AM for 30min, washed and stimulated with various concentration of SP (3pM-1µM). [Ca²⁺]_i response was calculated as percentage of 1µM ATP stimulation at endogenous purinergic receptors. Expression of NK1R protein was assessed by Western blot using an antibody against the N-terminus sequence (Novus Biologicals). Membranes and total lysate of transfected cells (20 and 40µg protein respectively) or membranes of stably transfected hNK1-CHO cells (40µg protein) were loaded onto 10% SDS-PAGE and proteins transferred to a PVDF membrane. Immunoreactive products were detected using ECL-Plus.

RT-PCR experiments from two independent transfections in COS-7 cells confirmed the transcription of both NK1R isoforms, whereas no amplification was obtained in mock-transfected cells. In binding experiments, cells transfected with the long isoform showed the presence of receptor sites ranging from 26 to 224fmol protein/100,000 cells in COS-7 and from 6 to 15fmol protein/100,000 cells in CHO-K1 (n=3). Accordingly, in transfected CHO-K1 cells, SP gave a maximal increase in [Ca²⁺]_i equal to 17.7±2.3% of ATP and showed a pEC₅₀ of 9.5±0.3 (mean±SEM of n=3). By contrast, cells transfected with the short NK1R did not show either [³H]-SP specific binding or a [Ca²⁺]_i response (n=3). Immunoblot analysis demonstrated a band of about 50kDa in membranes of both COS-7 cells transfected with the long NK1R and in stably transfected hNK1-CHO cells. Conversely, in cells tranfected with the short isoform, no immunoreactive product was observed in membranes, while in the total cell lysate a specific band was detected at approximately 35kDa, in agreement with the expected short NK1R molecular weight.

In conclusion, binding, functional and immunoblot experiments failed to detect the expression of the short hNK1R in membranes under the same assay conditions in which the long isoform was shown to be functionally expressed. This may indicate a different post-translational targeting of the two isoforms in these cell lines, which may influence their binding and/or functional properties.

Caberlotto L. et. al. (2003) Eur. J. Pharmacol 17: 1736-1746.
Fong T.M. et al.(1991) Mol. Pharmacol. 41: 24-30.