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The recent development of techniques for recording currents through single ionic channels has led to the identification of a K+-specific channel that is activated by cytoplasmic Ca2+. The channel has complex properties, being activated by depolarizing voltages and having a voltage-sensitivity that is modulated by cytoplasmic Ca2+ levels. The conduction behaviour of the channel is also unusual, its high ionic selectivity being displayed simultaneously with a very high unitary conductance. Very little is known about the biochemistry of this channel, largely due to the lack of a suitable ligand for use as a biochemical probe for the channel. We describe here a protein inhibitor of single Ca2+-activated K+ channels of mammalian skeletal muscle. This inhibitor, a minor component of the venom of the Israeli scorpion, Leiurus quinquestriatus, reversibly blocks the large Ca2+-activated K+ channel in a simple biomolecular reaction. We have partially purified the active component, a basic protein of relative molecular mass (Mr) approximately 7,000.

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The cold and menthol receptor, TRPM8, also designated CMR1, is a member of the transient receptor potential (TRP) family of excitatory ion channels. TRPM8 is a channel activated by cold temperatures, voltage, and menthol. In this study, we characterize the coldand voltage-induced activation of TRPM8 channel in an attempt to identify the temperature-and voltage-dependent components involved in channel activation. Under equilibrium conditions, decreasing temperature has two effects. Since the breakthrough identification of the vanilloid receptor, TRPV1, as the hot-capsaicin receptor (1), six temperature-dependent channels have been cloned during the last 7 years (1-8). Interestingly, all of the cloned channels belong to the extended transient receptor potential (TRP) family of excitatory channels (9, 10). TRP channels are membrane proteins constituted by subunits containing six-transmembrane domains, which assemble into tetramers forming conducting pores (11, 12). Heat-dependent channels belong to the vanilloid receptor TRPV subfamily and the cold-dependent channel TRPM8 belongs to the TRPM subfamily (13). Thermo-TRP channels have different temperature thresholds for activation, and their activity is modulated by several different agonists, allowing us to sense and differentiate a large spectrum of temperatures, from below 0°C to 50°C (14). Among those temperature-activated channels, TRPM8 was the first found to sense a cold stimulus (4, 7). TRPM8 is a nonselective outwardly rectifying channel. The channel opens at Ϸ28°C, and its activity increases as the temperature diminishes and saturates at Ϸ10°C (4). The channel is also able to respond to several agonists, including menthol (15).Channel gating is known to be affected by temperature. Q 10 value is used to estimate the temperature dependence of a given system. Channels with a Q 10 Ն 2 are considered highly temperature-dependent (16). Thermo-TRP receptor channels present exceptionally high Q 10 values (Ͼ20) (13).Several studies have addressed capsaicin binding (17, 18), pH dependencies (19), physiological relevance (13), and, recently, thermodynamics (20) of TRPV1, one of the molecular transducers of heat sensation. Large values for transitional entropy, enthalpy, and Q 10 were found, (20) indicating large rearrangements in the channel structure during activation. In contrast, little is known about regulation of its counterpart, TRPM8.Here we show that the large changes in TRPM8 channel gating induced by temperature are mainly due to modifications of the maximum probability of opening and to a shift along the voltage axis of the conductance-voltage curves. Moreover, the results can be fully explained by using an allosteric model in which temperature has only a moderate effect on the voltage sensors (Q 10 Ϸ 3) when channels are closed. Thus, temperature and voltage sensor activation act almost independently to promote channel opening. MethodsTemperature Control and Ramps. The recording chamber consisted of an electrically isolated bronze block with a hole of the...

Maxi-K channels consist of a pore-forming alpha subunit and a regulatory beta subunit, which confers the channel with a higher Ca(2+) sensitivity. Estradiol bound to the beta subunit and activated the Maxi-K channel (hSlo) only when both alpha and beta subunits were present. This activation was independent of the generation of intracellular signals and could be triggered by estradiol conjugated to a membrane-impenetrable carrier protein. This study documents the direct interaction of a hormone with a voltage-gated channel subunit and provides the molecular mechanism for the modulation of vascular smooth muscle Maxi-K channels by estrogens.

Ca2+-activated K+ channels of large conductance (MaxiK or BK channels) control a large variety of physiological processes, including smooth muscle tone, neurosecretion, and hearing. Despite being coded by a single gene (Slowpoke), the diversity of MaxiK channels is great. Regulatory b-subunits, splicing, and metabolic regulation create this diversity fundamental to the adequate function of many tissues.

The gating kinetics of a Cat'-activated K' channel from adult rat muscle plasma membrane are studied in artificial planar bilayers . Analysis of single-channel fluctuations distinguishes two Ca 2 '-and voltage-dependent processes : (a) short-lived channel closure (<1 ms) events appearing in a bursting pattern ; (b) opening and closing events ranging from one to several hundred milliseconds in duration . The latter process is studied independently of the first and is denoted as the primary gating mode .