E1678 Sheynin et al. with either of the mutated Nav1.7 subunits plus β1β2, exhibited currents that were not significantly different from those recorded from control cells (Figure 3). The mean peak currents at -20 mV were: -11 ± 3 pA/pF (n=7, p>0.6 vs control) and -13 ± 5 pA/pF (n=5, p>0.9 vs control) for Nav1.7-R896Q and Nav1.7-ΔR1370-L1374 respectively. Hence, the two mutations completely abolish the function of the voltage-gated sodium channel, which is consistent with the complete insensitivity to pain phenotype seen in affected individuals from these two families. DISCUSSION The ability to treat pain will hopefully improve upon our understanding of its molecular basis. The Nav1.7 voltage-gated sodium channel is a major and non-redundant part of pain perception and the identification of mutations that change its function holds the potential to contribute to pain relief. In particular, identification of the structural regions that are critical for the normal function of Nav1.7 is important as these regions could be targeted for the development of novel analgesics. However, all the CIP mutations reported to date have been truncating mutations, thus failing to reveal specific amino acids that are critical for normal function of Nav1.7. Here we present the first CIP missense and in-frame deletions in SCN9A. These mutations highlight specific functionally significant amino acids, both being in the same functional domain of the Nav1.7 protein. Previously reported missense mutations in Nav1.7 have been associated with PE, PEPD or febrile seizures and are mostly located to cytoplasmic linkers of the sodium channel, with some PE causing mutations in transmembrane domains. While it is not clear how and why these different gain-of-function mutations result in distinct disorders, it seems that PE causing mutations typically cause a hyperpolarizing shift in the voltage dependence of channel activation and a delay in deactivation of active channels upon return to non-activating membrane voltages. Both would increase the Nav1.7 currents at small deviations from the resting potential and the likelihood of action potential initiation, presumably sensitizing the neurons expressing these channels to small depolarising stimuli. PEPD causing mutations by contrast typically result in depolarizing shifts in the voltage dependence of fast inactivation, which is also seen in some PE-causing mutations, but importantly also rendering this process incomplete, thus resulting in persistent inward currents again leaving nociceptive neurons expressing these channels hyperexcitable [Drenth and Waxman, 2007]. More recently a mutation (Nav1.7-A1632E) which results in hyperpolarising shift in the voltage dependence of activation concomitantly with partial fast inactivation has been described in a patient with symptoms of both PE and PEPD [Estacion et al., 2008]. The missense mutation and in-frame deletion described here both map to the central sodium influx pore region of Nav1.7. The pore is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. The P segments are accessible only from the extracellular surface, with each undergoing a hairpin turn in the permeation pathway such that amino acids on both sides of the putative selectivity ﬁlter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial potassium channels to mammalian sodium channels identiﬁes this structure as a critical feature in the architecture of ion selective pores [Yamagishi et al., 2001]. The mutations we describe both cause a significant reduction in the number of cells with plasma membrane staining of Nav1.7 by immunocytochemistry in transfected PC12 cells. This is similar to what happens for specific mutations in SCN5A, where the mutant channel becomes retained in the endoplasmic reticulum (ER) [Liu et al., 2005; Baroudi et al., 2001]. It is therefore conceivable that the SCN9A mutations described here also cause misfolding of the channel which leads to ER retention and hence defective cell surface localization of Nav1.7. However, as a minority of cells overexpressing the mutant protein appeared to show some plasma membrane staining, it seems reasonable to hypothesize that even if some mutant protein can make it to the membrane, insufficient current densities are reached, possibly due to malfolding of the channel pore. Whichever is the principal mutational mechanism, the result is a non-functional sodium channel causing congenital insensitivity to pain in the patients. In summary, we have identified two novel non-truncating mutations in SCN9A which further expands the spectrum of mutations seen in Channelopathy-associated Insensitivity to Pain. The location of the mutations within the channel pore highlights the importance of this structure to channel function and shows how the highly regulated folding of this region can be disrupted by the alteration of a single amino acid. For analgesic design it suggests further possibilities: design of a moiety that can bind to the Nav1.7 sodium pore; and discovery, and then inhibition, of the chaperone protein that correctly folds Nav1.7 prior to its trafficking to the membrane.