Functional and molecular characterization of TRP channels in smooth muscle

Nonselective cation channels (NSCC) are targets of excitatory agonists in smooth muscle, representing I_cat. Na+ influx through NSCC causes depolarizations and activates voltage-dependent calcium channels, resulting in contraction. The molecular identity of I_cat in smooth muscle has not been elucidated, however, products of the transient receptor potential (TRP) genes have characteristics similar to native I_cat. We have determined the levels of TRP transcriptional expression in several murine and canine gastrointestinal (GI) and vascular smooth muscles and analyzed the alternative processing of these transcripts. Of the seven TRP gene family members, transcripts for TRP4, TRP6 and TRP7 were detected in all murine and canine smooth muscle cell preparations. The full-length cDNAs for TRP4, TRP6 and TRP7, as well as one splice variant of TRP4 and two splice variants of TRP7, were isolated from murine colonic smooth muscle. Quantitative RT-PCR determined the relative amounts of TRP4, TRP6 and TRP7 transcripts, as well as that of the splice variants, in several murine smooth muscles. TRP4 is the most highly expressed, while TRP6 and TRP7 are expressed at a lower level in the same tissues.; Interstitial cells of Cajal (ICC) are the pacemaker cells responsible for the generation and propagation of electrical slow waves in phasic muscles of the gastrointestinal (GI) tract. The pacemaker current that initiates each slow wave derives from a calcium-inhibited, voltage independent nonselective cation channel. This channel in ICC displays properties similar to that reported for the Transient Receptor Potential (TRP) family of non-selective cation channels, particularly those seen for TRPC3 and TRPC4. We have identified transcripts for TRPC4 in individually isolated ICC and have cloned the two alternatively spliced forms of TRPC4; TRPC4α and TRPC4β from GI muscles. TRPC4β is missing an 84 amino acid segment from the carboxy terminus. Expression of either form using the whole-cell patch clamp technique led to calcium-inhibited nonselective cation channels as determined by NMDG + replacement experiments and BAPTA dialysis. Expression of TRPC4β channels recorded at the whole cell level had characteristics similar to the nonselective cation current in ICC. The single channel conductance of TRPC4β was determined to be 17.5pS. Application of calmidazolium to cells expressing TRPC4β led to a significant increase in the inward current of these cells at both the whole cell and single channel level, and currents were sensitive to block by 10μM lanthanum, niflumic acid and DIDS. Comparison of the properties reported for the nonselective cation current in ICC and those identified here for TRPC4β led us to conclude that a TRPC4-like current encodes the plasmalemmal pacemaker current in murine small intestine.