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Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons. Voltage-gated sodium channels function as multiprotein signaling complexes. Here, authors show that the dispanin TMEM233 is essential for activity of stinging nettle toxins and that co-expression of TMEM233 modulates the gating properties of NaV1.7. [ABSTRACT FROM AUTHOR]

Voltage-dependent calcium ([Ca.sup.2+], [Ca.sub.v]1.2) channels are the primary [Ca.sup.2+] entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified [Ca.sub.v]1.2 [[alpha].sub.1]-subunit splice variation in myocytes of rat resistance-size (100-200 [micro]m diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon 1c exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and [+ or -] 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major [Ca.sub.v]1.2 variant contained exons 1c, 8, 21, and 32+33, with ~57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon 1b/c/ [+ or -] 9* variants were characterized following transfection in HEK293 cells. Exon 1c and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon 1b, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltage-dependent inactivation. In summary, we have identified the [Ca.sub.v]1.2 [[alpha].sub.1]-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell [Ca.sub.v]1.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range. voltage-dependent calcium channel; myogenic artery; cloning; ribonucleic acid splicing

A capacitive micromachined ultrasonic transducer (CMUT) array for minimally invasive medical diagnosis has been developed. Unlike traditional ultrasonic transducers, which generally use a bulky piece of substrate, this transducer array was integrated on a 40-[micro]m-thick micromachined silicon substrate into a probe shape with a typical shank width of 50-80 [micro]m and a shank length of 4-8 mm. For 1-D arrays, 24-96 CMUT devices were integrated on one such silicon probe and formed an accurately configured phase array. In addition to miniaturization, reduction of the substrate thickness also decreases the intertransducer crosstalk due to substrate Lamb waves. Due to its miniature size, this array can be placed or implanted close to the target tissue/organ and can perform high-resolution high-precision diagnosis and stimulation using high-frequency ultrasounds. The issue of conflict between resolution and penetration depth of ultrasonic diagnosis can therefore be resolved. A two-layer polysilicon surface micromachining process was used to fabricate this device. Suspended polysilicon membranes of diameters ranging from 20 to 90 [micro]ms and thicknesses from 1.0 to 2.5 [micro]m were used to generate and detect ultrasounds of frequencies ranging from 1 to 10 MHz. B-mode imaging using this transducer array has been demonstrated. [2007-0125] Index Terms--Acoustic devices, acoustic transducers, actuators.

This paper presents the first monolithic multidirection-looking ultrasonic imager for minimally invasive medical diagnosis. In contrast to the traditional planar ultrasonic imagers that can only view in one direction, this 3-D array is able to view in multiple directions by using seven planar imagers integrated on a hexagonal silicon prism. Each facet on the prism is integrated with a planar 1- or 2-D capacitive-micromachined ultrasonic-transducer imager array for viewing in a specific direction. Each facet is connected by a flexible dielectric membrane, which is monolithically fabricated with the transducers. The dielectric membranes also support the thin-film electrical interconnects between the arrays on different facets. The substrate is folded into a hexagonal prism after completion of the transducer microfabrication process. With this architecture, a one flip-chip bonded or monolithically integrated front-end electronic circuit will be able to manage all the imagers on the 3-D array. The number of bonding wires for a connection to external electronics can therefore be reduced. Imager prisms, which are ranging from 1 to 4 mm in diameter and 2 to 4 mm in length, and positioned to view in seven directions, have been prototyped. Preliminary testing shows that the imager transducers behaved consistently before and after the assembly process. Applications of this 3-D imager array include capsule ultrasound endoscope, intravascular ultrasound, and other internal imaging needs. [2006-0269] Index Terms--Acoustic transducers.

L-type, voltage-dependent calcium ([Ca.sup.2+]) channels, ryanodine-sensitive [Ca.sup.2+] release (RYR) channels, and large-conductance [Ca.sup.2+] activated potassium ([K.sub.Ca]) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters [Ca.sup.2+] spark to [K.sub.Ca] channel coupling and [Ca.sup.2+] spark regulation by voltage-dependent [Ca.sup.2+] channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control ([cav-1.sup.+/+] cells but were not observed in cav-1-deficient ([cav-1.sup.-/-]) cells. [Ca.sup.2+] spark and transient [K.sub.Ca] current frequency were approximately twofold higher in [cav-1.sup.-/-] than in [cav-1.sup.+/+] cells. Although voltage-dependent [Ca.sup.2+] current density was similar in [cav-1.sup.+/+] and [cav.sup.-1-/-] cells, diltiazem and [Cd.sup.2+], voltage-dependent [Ca.sup.2+] channel blockers, reduced transient [K.sub.Ca] current frequency to ~55% of control in [cav-1.sup.+/+] cells but did not alter transient [K.sub.Ca] current frequency in [cav-1.sup.-/-] cells. Furthermore, although [K.sub.Ca] channel density was elevated in [cav-1.sup.-/-] cells, transient [K.sub.Ca] current amplitude was similar to that in [cav-1.sup.+/+] cells. Higher [Ca.sup.2+] spark frequency in [cav-1.sup.-/-] cells was not due to elevated intracellular [Ca.sup.2+] concentration, sarcoplasmic reticulum [Ca.sup.2+] load, or nitric oxide synthase activity. Similarly, [Ca.sup.2+] spark amplitude and spread, the percentage of [Ca.sup.2+] sparks that activated a transient [K.sub.Ca], current, the amplitude relationship between sparks and transient [K.sub.Ca] currents, and [K.sub.Ca] channel conductance and apparent [Ca.sup.2+] sensitivity were similar in [cav-1.sup.+/+] and [cav-1.sup.-/-] cells. In summary, cav-1 ablation elevates [Ca.sup.2+] spark and transient [K.sub.Ca] current frequency, attenuates the coupling relationship between voltage-dependent [Ca.sup.2+] channels and RyR channels that generate [Ca.sup.2+] sparks, and elevates [K.sub.Ca] channel density but does not alter transient [K.sub.Ca] current activation by [Ca.sup.2+] sparks. These findings indicate that cav-1 is required for physiological [Ca.sup.2+] spark and transient [K.sub.Ca] current regulation in cerebral artery smooth muscle cells. ryanodine-sensitive [Ca.sup.2+] release channel; large-conductance [Ca.sup.2+]-activated potassium channel; caveolae; voltage-dependent [Ca.sup.2+] channel

Abstract: Gain-of-function and loss-of-function mutations in Nav1.7 cause chronic pain and pain insensitivity, respectively. The preferential expression of Nav1.7 in the peripheral nervous system and its role in human pain signaling make Nav1.7 a promising target for next-generation pain therapeutics. However, pharmacological agents have not fully recapitulated these pain phenotypes, and because of the lack of subtype-selective molecular modulators, the role of Nav1.7 in the perception of pain remains poorly understood. Scorpion venom is an excellent source of bioactive peptides that modulate various ion channels, including voltage-gated sodium (Nav) channels. Here, we demonstrate that Buthus martensii Karsch scorpion venom (BV) elicits pain responses in mice through direct enhancement of Nav1.7 activity and have identified Makatoxin-3, an α-like toxin, as a critical component for BV-mediated effects on Nav1.7. Blocking other Nav subtypes did not eliminate BV-evoked pain responses, supporting the pivotal role of Nav1.7 in BV-induced pain. Makatoxin-3 acts on the S3-S4 loop of voltage sensor domain IV (VSD4) of Nav1.7, which causes a hyperpolarizing shift in the steady-state fast inactivation and impairs inactivation kinetics. We also determined the key residues and structure-function relationships for the toxin-channel interactions, which are distinct from those of other well-studied α toxins. This study not only reveals a new mechanism underlying BV-evoked pain but also enriches our knowledge of key structural elements of scorpion toxins that are pivotal for toxin-Nav1.7 interactions, which facilitates the design of novel Nav1.7 selective modulators. [ABSTRACT FROM AUTHOR]

Abstract Background Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals. Results Small diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro. Conclusions NCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

Abstract Background Two groups of gain-of-function mutations in sodium channel NaV1.7, which are expressed in dorsal root ganglion (DRG) neurons, produce two clinically-distinct pain syndromes - inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). IEM is characterized by intermittent burning pain and skin redness in the feet or hands, triggered by warmth or mild exercise, while PEPD is characterized by episodes of rectal, ocular and mandibular pain accompanied with skin flushing, triggered by bowel movement and perianal stimulation. Most of the IEM mutations are located within channel domains I and II, while most of the PEPD mutations are located within domains III and IV. The structural dichotomy parallels the biophysical effects of the two types of mutations, with IEM mutations shifting voltage-dependence of NaV1.7 activation in a hyperpolarized direction, and PEPD mutations shifting fast-inactivation of NaV1.7 in a depolarized direction. While four IEM and four PEPD mutations are located within cytoplasmic linkers joining segments 4 and 5 (S4-S5 linkers) in the different domains (IEM: domains I and II; PEPD: domains III and IV), no S4-S5 linker has been reported to house both IEM and PEPD mutations thus far. Results We have identified a new IEM mutation P1308L within the C-terminus of the DIII/S4-S5 linker of NaV1.7, ten amino acids from a known PEPD mutation V1298F which is located within the N-terminus of this linker. We used voltage-clamp to compare the biophysical properties of the two mutant channels and current-clamp to study their effects on DRG neuron excitability. We confirm that P1308L and V1298F behave as prototypical IEM and PEPD mutations, respectively. We also show that DRG neurons expressing either P1308L or V1298F become hyperexcitable, compared to DRG neurons expressing wild-type channels. Conclusions Our results provide evidence for differential roles of the DIII/S4-S5 linker N- and C-termini in channel inactivation and activation, and demonstrate the cellular basis for pain in patients carrying these mutations.

Abstract Background Primary erythromelalgia is an autosomal dominant pain disorder characterized by burning pain and skin redness in the extremities, with onset of symptoms during the first decade in the families whose mutations have been physiologically studied to date. Several mutations of voltage-gated Na+ channel NaV1.7 have been linked with primary erythromelalgia. Recently, a new substitution NaV1.7/I136V has been reported in a Taiwanese family, in which pain appeared at later ages (9–22 years, with onset at 17 years of age or later in 5 of 7 family members), with relatively slow progression (8–10 years) to involvement of the hands. The proband reported onset of symptoms first in his feet at the age of 11, which then progressed to his hands at the age of 19. The new mutation is located in transmembrane segment 1 (S1) of domain I (DI) in contrast to all NaV1.7 mutations reported to date, which have been localized in the voltage sensor S4, the linker joining segments S4 and S5 or pore-lining segments S5 and S6 in DI, II and III. Results In this study, we characterized the gating and kinetic properties of I136V mutant channels in HEK293 cells using whole-cell patch clamp. I136V shifts the voltage-dependence of activation by -5.7 mV, a smaller shift in activation than the other erythromelalgia mutations that have been characterized. I136V also decreases the deactivation rate, and generates larger ramp currents. Conclusion The I136V substitution in NaV1.7 alters channel gating and kinetic properties. Each of these changes may contribute to increased excitability of nociceptive dorsal root ganglion neurons, which underlies pain in erythromelalgia. The smaller shift in voltage-dependence of activation of NaV1.7, compared to the other reported cases of inherited erythromelalgia, may contribute to the later age of onset and slower progression of the symptoms reported in association with this mutation.