Pedro Labarca

A ciliary K+ conductance sensitive to charibdotoxin underlies inhibitory responses in toad olfactory receptor neurons.

In olfactory neurons from Caudiverbera caudiverbera, a mixture of putrid odorants trigger an inhibitory, K+‐selective current and a hyperpolarizing receptor potential. The current‐voltage relation resembles that of a Ca2+‐activated K+ conductance; their amplitude depends on extracellular Ca2+. 10 nM charibdotoxin, a blocker of K+‐selective channels, including Ca2+‐activated ones, reversibly abolished inhibitory currents and receptor potentials. Focal stimulation demonstrates that the underlying transduction mechanism is confined to the cilia. This represents the first evidence for inhibitory responses in vertebrate olfactory cells mediated by a ciliary CTX‐sensitive K+ conductance, most likely a Ca2+‐activated one.

Pore accessibility during C‐type inactivation in Shaker K+ channels

Shaker K+ channels inactivate through two distinct molecular mechanisms: N‐type, which involves the N‐terminal domain and C‐type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C‐type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C‐type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C‐type inactivated channels. The structural changes Shaker channels undergo during C‐type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.

SELECTIVITY AND GATING PROPERTIES OF A CAMP-MODULATED, K+-SELECTIVE CHANNEL FROM DROSOPHILA LARVAL MUSCLE

The selectivity and gating properties of cAMP‐modulated, voltage‐independent, K+‐selective channel from Drosophila larval muscle were investigated using the patch‐clamp technique. In symmetrical 115 mM K+ the channel displayed a linear current‐voltage relation with slope conductance of 43 pS. Under biionic conditions (115 mM K+ pipette/115 mM X+ cytoplasmic) the permeability sequence was K+>Rb+>NH+ 4 ⪢ Cs+,Na+. The channel was impermeable to Ca2+ (P Ca/P K < 0.02). Under steady‐state conditions and regardless [cAMP], open dwell times showed a double exponential distribution. [cAMP] did not affect the time constants of the two components of open times, or their relative amplitudes. Moreover, successive openings were correlated in open time. Closed dwell times were made of at least three exponential components. Fast application of cAMP to the cytoplasmic side of the channel induced a transient increase in open probability that relaxed to a lower value within seconds. This last result suggests that cAMP can activate and desensitize this cAMP‐modulated, K+‐selective channel.

Inhibitory Responses to Odorants in Vertebrate Olfactory Neurons

In vertebrates, the primary steps in olfaction take place at the olfactory epithelium. In this neuroepithelium, bipolar sensory olfactory neurons transduce chemical stimuli into electrical signals. Olfactory neurons project from their apical pole, towards the epithelial surface, a dendrite ending in a knob, to which a variable number of olfactory cilia are attached. Unmyelinated axons, originating from the basal pole of olfactory neurons, reach the olfactory bulb to make contact with secondary cells. Typically, olfactory neurons exhibit spontaneous action potential firing. Exposure to odorants gives place to changes in action potential firing rates.

Native and chemically modified porin channels from Salmonella typhi Ty2 in planar lipid bilayers

Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Plp) were studied in planar lipid bilayers. The conductance of bilayers exposed to native or chemically modified porins increases in discrete jumps. Conductance histograms for native porins displayed two major peaks at 1.7 and 6.7 nS (in 0.5 M KCl). On the other hand, Plp‐treated porins exhibited a single major peak at 1 nS. The relation between bilayer conductance and native porin concentration was linear. However, this relation became logarithmic in the presence of modified porins. The results support the notion that alkaline reduction of S. typhi Ty2 porins with Plp dissociates porin channel trimers in a reversible fashion.

POSSIBLE PARTICIPATION OF A cAMP REGULATED K+ CHANNEL FROM THE SEA URCHIN SPERM IN THE SPERACT RESPONSE

Ion channels are key elements in the activation of respiration and motility, in chemotaxis, and in triggering the acrosome reaction (AR) in sperm (Schackmann, 1989; Ward and Kopf, 1993; Darszon et al., 1994). The small size of these cells (head diameter ∼ 2 μm) has precluded the careful characterization of their electrophysiological properties. Because of this we have used planar bilayers with incorporated sperm plasma membrane components, to detect single channels from sea urchin sperm. Although with a low success rate we have also succeeded in patch clamping sea urchin sperm (Guerrero et al., 1986; Babcock et al., 1992). Using these techniques, together with studies of membrane potential, [Ca2+]i and pHi in whole sperm, we have established the presence of K+, Ca2+ and Cl- channels in this cell and are exploring their regulation and participation in chemotaxis and in the AR (Darszon et al., 1994).

Ion channels: Key elements in sea urchin sperm physiology

Ion channels are deeply involved in sea urchin sperm activation, motility, chemotaxis and in the acrosome reaction. Unraveling ion channel function and regulation in sperm behavior has required a combination of complementary approaches since spermatozoa are very tiny cells. Planar bilayer and patch clamp techniques have allowed us to detect, for the first time, the activity of single channels in the plasma membrane of these cells. Unlike intact sperm, swollen sperm can be much more easily patch clamped and single channel activity recorded. These techniques, together with studies of membrane potential, intracellular Ca2+ and pH in whole sperm, have established the presence of K+, Ca2+, and Cl− channels in this cell. The strategies developed to study sea urchin sperm channels are applicable to mammalian spermatozoa. We recently detected a Ca2+ channel resembling one found in S. purpuratus sperm in planar bilayers containing mouse sperm plasma membranes. The presence of this Ca2+ channel in such diverse spec...

Gating and Selectivity Properties of a cAMP-Activated K+ Selective Channel from Drosophila Larval Muscle

For a while, direct modulation of ion channels by cyclic nucleotides was thought to be confined to those cation-selective ones found in photoreceptors and in vertebrate olfactory neurons (Kaupp, 1991; Zufall, 1994). This idea was fortified by structural and functional similarities exhibited by cyclic nucleotide-gated channels in olfactory neurons with those of photoreceptor cells. The presence of a ciliary axonema in photoreceptors cells pointed out to a unique evolutionary relationship between visual and olfactor y sensory cells. However, cyclic nucleotide activated cation-selective channels have been reported now in a variety of animal cells, including retinal ganglion cells, aorta, testis, kidney, heart and sperm (Yau, 1994). Furthermore, there is increasing evidence for the presence in animals of K+-selective channels that are directly gated by cyclic nucleotides (Delgado et al., 1991; Michael and Asche, 1992; Bruggemann et al., 1993; Gotow, et al., 1994; Gomez and Nassi, 1995; Jorquera et al., 1995; Labarca et al., 1995).