Sacha A. Malin

TPRV1 Expression Defines Functionally Distinct Pelvic Colon Afferents

Changes in primary sensory neurons are likely to contribute to the emergence of chronic visceral pain. An important step in understanding visceral pain is the development of comprehensive phenotypes that combines functional and anatomical properties for these neurons. We developed a novel ex vivo physiology preparation in mice that allows intracellular recording from colon sensory neurons during colon distension, in the presence and absence of pharmacologic agents. This preparation also allows recovery of functionally characterized afferents for histochemical analysis. Recordings obtained from L6 dorsal root ganglion cells in C57BL/6 mice identified two distinct populations of distension-responsive colon afferents: high-firing frequency (HF) and low-firing frequency (LF) cells. Fluid distension of the colon elicited rapid firing (>20 Hz) in HF cells, whereas LF cells seldom fired >5 Hz. Distension response thresholds were significantly lower in HF cells (LF, 17.5 ± 1.1 cmH2O; HF, 2.6 ± 1.0 cmH2O). Responses of most LF afferents to colon distension were sensitized by luminal application of capsaicin (1 μm; 8 of 9 LF cells), mustard oil (100 μm; 10 of 12 LF cells), and low pH (pH 4.0; 5 of 6 LF cells). In contrast, few HF afferents were sensitized by capsaicin (3 of 9), mustard oil (2 of 7), or low pH (1 of 6) application. Few HF afferents (4 of 23) expressed the capsaicin receptor, TRPV1. In contrast, 87% (25 of 29) of LF afferents expressed TRPV1. TRPV1 has been shown to be required for development of inflammatory hyperalgesia. These results suggest a unique functional role of TRPV1-positive colon afferents that could be exploited to design specific therapies for visceral hypersensitivity.

Presenilins Upregulate Functional K+Channel Currents in Mammalian Cells☆

Mutations in presenilin 1 (PS-1) and presenilin 2 (PS-2) have been linked to early onset, autosomal dominant Alzheimers disease. Neither the normal function(s) of the presenilins nor their role(s) in mediating the devastating neurological and pathological changes associated with Alzheimers Disease, however, are well understood. The results of the experiments described here demonstrate that expression of wild-type PS-1 or PS-2 increases outward K+ current densities in HEK-293 cells relative to untransfected or mock-transfected cells. Western blot analysis reveals that there is a marked increase in full-length, rather than processed, presenilins in transiently transfected HEK-293 cells, suggesting that full-length PS-1 (or PS-2) underlies the observed increases in outward K+ current densities. Consistent with this hypothesis, EXpression of an N-terminal proteolytic fragment of PS-1 is without effects on the membrane properties of HEK-293 cells. Mean outward K+ current densities are also shown to be increased in HEK-293 cells expressing the exon 9 splice site PS-1 mutation (deltaex9/PS-1), a mutant that does not undergo proteolytic processing. In HEK- 293 cells transiently transfected with a missense (G209V) PS-1 mutant, however, mean K+ current densities were not significantly different from controls. Expression of wild-type PS-1 in neonatal rat ventricular myocytes also results in increased outward K+ currents, whereas no detectable effects on membrane currents were seen in PS-1-transfected COS-7 cells. These results suggest that the presenilins do not actually form K+ channels, but rather that these proteins upregulate functional K+ channel expression either directly by associating with K+ channel pore-forming subunits or indirectly by increasing the synthesis, assembly, and/or transport of these subunits. The observation that PS-1 and PS-2 are highly expressed in neurons, localized to the endoplasmic reticulum, suggests that the presenilins could regulate neuronal K+ channel expression; mutations in PS-1/PS-2 would then be expected to result in profound changes in neuronal excitability and contribute to the cognitive decline commonly associated with Alzheimers Disease.